U.S. patent application number 12/546063 was filed with the patent office on 2009-12-17 for compositions for alleviating inflammation and oxidative stress in a mammal.
This patent application is currently assigned to LIFELINE NUTRACEUTICALS CORPORATION. Invention is credited to William J. Driscoll, Paul R. Myhill.
Application Number | 20090311350 12/546063 |
Document ID | / |
Family ID | 34968759 |
Filed Date | 2009-12-17 |
United States Patent
Application |
20090311350 |
Kind Code |
A1 |
Myhill; Paul R. ; et
al. |
December 17, 2009 |
COMPOSITIONS FOR ALLEVIATING INFLAMMATION AND OXIDATIVE STRESS IN A
MAMMAL
Abstract
An antioxidant-promoting composition that increases antioxidant
defense potential in a subject is disclosed comprising Bacopa
monniera extract; milk thistle extract, ashwagandha powder, green
tea extract, Gotu kola powder, Ginko biloba leaf extract; Aloe vera
powder; turmeric extract; and N-acetyl cysteine. The
antioxidant-promoting composition of the invention safely induces
cellular antioxidant potential to achieve an overall net decrease
in oxidative stress without the undesirable side-effects associated
with the individual components of the antioxidant-promoting
composition. Also disclosed is a method for reducing the
undesirable side-effects of free radicals in a subject by
administering to a subject in need of such antioxidants an
effective amount of antioxidant-promoting composition of the
invention.
Inventors: |
Myhill; Paul R.; (Castle
Rock, CO) ; Driscoll; William J.; (Castle Rock,
CO) |
Correspondence
Address: |
SWANSON & BRATSCHUN, L.L.C.
8210 SOUTHPARK TERRACE
LITTLETON
CO
80120
US
|
Assignee: |
LIFELINE NUTRACEUTICALS
CORPORATION
Littleton
CO
|
Family ID: |
34968759 |
Appl. No.: |
12/546063 |
Filed: |
August 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11216514 |
Aug 31, 2005 |
7579026 |
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12546063 |
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11088323 |
Mar 23, 2005 |
7241461 |
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11216514 |
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60555802 |
Mar 23, 2004 |
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60590528 |
Jul 23, 2004 |
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60604638 |
Aug 26, 2004 |
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60607648 |
Sep 7, 2004 |
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60610749 |
Sep 17, 2004 |
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60643754 |
Jan 13, 2005 |
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60646707 |
Jan 25, 2005 |
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Current U.S.
Class: |
424/725 |
Current CPC
Class: |
A61K 36/28 20130101;
A61P 17/02 20180101; A61P 19/02 20180101; A61K 38/446 20130101;
A61P 31/00 20180101; A61K 36/9066 20130101; A61P 9/10 20180101;
A61P 7/06 20180101; A61P 39/00 20180101; A61P 31/04 20180101; A61K
36/16 20130101; A61P 29/00 20180101; A61K 36/81 20130101; A61K
36/82 20130101; A61K 36/63 20130101; A61P 3/00 20180101; A61P 3/10
20180101; A61K 45/06 20130101; A61K 36/886 20130101; A61P 31/18
20180101; A61P 39/06 20180101; A61P 9/12 20180101; A61P 25/00
20180101; A61K 36/23 20130101; A61P 43/00 20180101; A61P 37/00
20180101; A61K 36/68 20130101; A61K 36/80 20130101; A61P 35/00
20180101; A61K 36/16 20130101; A61K 2300/00 20130101; A61K 36/23
20130101; A61K 2300/00 20130101; A61K 36/63 20130101; A61K 2300/00
20130101; A61K 36/80 20130101; A61K 2300/00 20130101; A61K 36/82
20130101; A61K 2300/00 20130101; A61K 36/886 20130101; A61K 2300/00
20130101; A61K 36/9066 20130101; A61K 2300/00 20130101; A61K 38/446
20130101; A61K 2300/00 20130101; A61K 36/28 20130101; A61K 2300/00
20130101; A61K 36/68 20130101; A61K 2300/00 20130101; A61K 36/81
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/725 |
International
Class: |
A61K 36/81 20060101
A61K036/81; A61P 29/00 20060101 A61P029/00 |
Claims
1. A composition comprising: (a) at least about 150 milligrams
Bacopa monniera extract, wherein said Bacopa monniera extract
having 20 percent bacosides or greater; at least about 225
milligrams Silybum marianum (milk thistle) extract, wherein said
milk thistle extract is having between about 70 percent and about
80 percent silymarin; at least about 150 mg Withania somnifera
(ashwagandha) powder; at least about 75 milligrams Camellia
sinensis (green tea) extract; wherein said green tea extract is
comprised of 98 percent polyphenols; said polyphenols comprised of
45 percent (-)-epigallocatechin gallate; at least about 75
milligrams Curcuma longa (turmeric) extract; wherein said turmeric
extract is comprised of 95 percent curcumin; wherein the
composition increases the enzyme activity level of at least one
antioxidant enzyme selected from the group consisting of:
superoxide dismutase and catalase and decreases the plasma
concentration level of thiobarbituric acid reactive chemical
species, when administered in an effective amount to a mammalian
subject in need thereof.
2. The composition according to claim 1, wherein the composition is
formulated as an oral dosage form.
3. The composition according to claim 2, wherein the oral dosage
form is selected from the group consisting of: a tablet; capsule;
and caplet.
4. The method of claim 1, wherein the composition includes one or
more excipients selected from the group consisting of calcium
carbonate; croscarmellose sodium; dicalcium phosphate; magnesium
stearate; microcrystalline cellulose; modified cellulose; silica;
and stearic acid.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/216,514, filed Aug. 31, 2005, which is a continuation of
U.S. application Ser. No. 11/088,323, filed Mar. 23, 2005, now U.S.
Pat. No. 7,241,461, U.S. Pat. No. 7,241,461 claims the benefit of
priority under 35 U.S.C. .sctn.120 from U.S. Application Ser. No.
60/555,802, filed on Mar. 23, 2004; U.S. Application Ser. No.
60/590,528, filed on Jul. 23, 2004; U.S. Application Ser. No.
60/604,638, filed on Aug. 26, 2004; U.S. Application Ser. No.
60/607,648, filed on Sep. 7, 2004; U.S. Application Ser. No.
60/610,749, filed on Sep. 17, 2004; U.S. Application Ser. No.
60/643,754, filed on Jan. 13, 2005, and U.S. Application Ser. No.
60/646,707, filed on Jan. 25, 2005, the contents of which are
incorporated herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions for
alleviating inflammation and oxidative stress in a subject.
Specifically, the present invention is directed to the field of
natural remedies and the development of compositions to increase
the antioxidant potential of a subject by inducing the subject's
natural cellular defenses.
DESCRIPTION OF THE INVENTION
[0003] Free radicals have come to be appreciated for their
importance to human health and disease. Many common and
life-threatening diseases, including atherosclerosis, cancer, and
aging, have free radical reactions as an underlying mechanism of
injury. One of the most common types of radicals is the reactive
oxygen species (ROS). These are the products of normal cell
respiration and metabolism and are generally regulated by cellular
defense systems present in the body. Such cellular defense systems
reduce the amount of damage that free and reactive species radicals
may cause by scavenging free radicals or enzymatically converting
the free radicals to less toxic chemical species, thereby serving a
physiological role as antioxidants.
[0004] Superoxide dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GPX) are three mammalian enzymes important to cellular
defense against ROS-mediated cellular damage. Due to environmental
agents such as pollution, and lifestyle factors such as smoking or
exercising, the cellular exposure to free radicals is increased.
Such increase may bring the body out of balance, especially as the
body ages and the mechanisms that produce antioxidants or remove
ROS are insufficient to counter oxidative stress. The body's
antioxidant defense system(s), including SOD, CAT, and GPX, can be
impaired by the aging process and or compromised, for example, by
inflammation, microbial infection, viral infection, the progression
of cancer or neurological disorders, and many other disorders
characterized by or caused in part by oxidative stress. The
resulting damage can range from disruption of biological processes,
killing of cells, and mutation of genetic material, which may lead
to the occurrence of cancer. Accordingly, the potential use of
antioxidant containing dietary supplements for protection against
the effects of oxidative stress and the progression of degenerative
diseases and aging has been the subject of an increasing number of
studies during the past four decades, see for example, Pauling L.,
N Engl J Med., Vitamin C therapy of advanced cancer, Mar. 20, 1980;
302(12):694-5.
[0005] Non-enzymatic antioxidants can react with free radicals
directly and become self-oxidized (therefore no longer available to
quench free radicals); or one antioxidant may act as a reducing
agent and another antioxidant oxidized in cyclical fashion (e.g.,
the interaction of ascorbic acid and alpha-tocopherol). Other
non-enzymatic free radical scavengers have been used experimentally
with varying results (e.g. mannitol, PBS, etc.); their clinical use
is severely limited due to their toxicities. Other synthetic
antioxidants, e.g., BHA (butylated hydroxy anisole), BHT (butylated
hydroxy toluene) and NDGA (nordihydro guaiaretic acid) may cause
allergic reactions and oncogenesis due to their strong toxicity in
the body, and be easily disrupted by heat due to temperature
sensitivity.
[0006] Enzymatic antioxidants (e.g., SOD, CAT and GPX), on the
other hand, are not consumed in the reactions with free radicals,
although they can be damaged under pathological conditions and
consequently rendered non-functional. In the local cellular milieu,
damaged enzymatic antioxidants would render that cellular
environment compromised and subject to free radical attack. The
disadvantage of administering enzymatic antioxidants to humans is
(1) the possibility of allergic reactions (in the case of a
bacterial or fungal derived enzyme) of varying degrees of severity;
(2) the great cost of harvesting these enzymes; (3) the limitation
of quantities of enzymatic antioxidants able to be administered at
a given time (theoretically to avoid side-effects such as serum
sickness or other immune reaction to the recombinant protein); (4)
they serve a singular purpose (i.e., they react with only one type
of oxidant); and (5) they do not quench all radicals, that may be
important for beneficial metabolic pathways, e.g., nitric oxide
induced vasodilation and immune system support (see, e.g.,
Griscavage J M, Wilk S, Ignarro L J., Proc Natl Acad Sci USA.,
Inhibitors of the proteasome pathway interfere with induction of
nitric oxide synthase in macrophages by blocking activation of
transcription factor NF-kappa B., Apr. 16, 1996; 93(8):3308-12.
[0007] The efficacy of direct oral administration of SOD, CAT, or
GPX has been limited by the sensitivity of these enzymes to the
milieu of the digestive system and/or lack of bioavailability. For
example, research indicated that the digestive system destroyed
SOD, and that neither CAT nor GPX was absorbed via the digestive
tract.
[0008] Since administration by direct ingestion of the antioxidants
showed disappointing results, efforts have been directed to provide
the body with the so-called "building blocks" of each of the three
antioxidants. A promising approach has been the development of
compositions directed toward increasing the activities of SOD, CAT,
and GPX in the body. Accordingly, supplements have been formulated
to increase levels of the body's zinc, copper, and manganese, to
assist the body's production of SOD. Similarly, iron, selenium, and
glutathione related supplements have been developed to increase CAT
and GPX. These compounds have toxic effects in large
quantities.
SUMMARY OF THE INVENTION
[0009] The present invention provides compositions that can be
administered to a mammalian subject, and will safely induce in the
subject, increased antioxidant potential in the subject by
increasing the activity of at least one antioxidant enzyme which
include, e.g., SOD, CAT, and GPX, and thereby decreasing the tissue
level of pathologic free radical species. The compositions of the
present invention yield an overall net decrease in oxidative stress
and inflammation when administered to a mammalian subject in an
effective amount with minimal undesirable side-effects.
Advantageously, the compositions of the invention provides fewer
side effects than may be associated with each of the individual
active agents in the composition.
[0010] In one aspect, the invention provides an
antioxidant-promoting composition that comprises active ingredients
comprising at least two agents that include, e.g., Bacopa monniera
extract; milk thistle extract, ashwagandha powder, green tea
extract, Gotu kola powder, Ginko biloba leaf extract; Aloe vera
powder; turmeric extract; and N-acetyl cysteine, wherein the
composition increases the enzyme activity level of at least one
antioxidant enzyme, e.g., superoxide dismutase; catalase; and
glutathione peroxidase and decreases the plasma concentration level
of thiobarbituric acid reactive chemical species, when administered
in an effective amount to a mammalian subject in need thereof. In
one embodiment, the Bacopa monniera extract is standardized extract
prepared from the leaves of the Bacopa monniera plant. In one
embodiment, the Bacopa monniera standardized extract contains at
least 20% bacosides. In one embodiment, the Bacopa monniera extract
is present at a concentration of about 5 weight percent to about 50
weight percent of the total dry weight of active ingredients of the
composition. In one embodiment, the Bacopa monniera extract is
present at a concentration of about 10 weight percent to about 30
weight percent of the total dry weight of active ingredients of the
composition. In one embodiment, the Bacopa monniera extract is
present at a concentration at least about 22 weight percent of the
total dry weight of active ingredients of the composition. In one
embodiment, Bacopa monniera extract is present at a concentration
at least about 12 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the milk thistle
extract is standardized to contain at least about 70 weight percent
silymarin. In one embodiment, the milk thistle extract is present
at a concentration from about 5 weight percent to about 60 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the milk thistle extract is present
at a concentration from about 10 weight percent to about 50 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the milk thistle extract is present
at a concentration at least about 33 weight percent of the total
dry weight of active ingredients of the composition. In one
embodiment, the milk thistle extract is present at a concentration
at least about 22 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the ashwagandha
powder is present at a concentration from about 5 weight percent to
about 50 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the ashwagandha
powder is present at a concentration from about 10 weight percent
to about 30 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the ashwagandha
powder is present at a concentration at least about 22 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the ashwagandha powder is present
at a concentration at least about 12 weight percent of the total
dry weight of active ingredients of the composition. In one
embodiment, the turmeric extract is standardized to contain at
least about 95 weight percent curcumin. In one embodiment, turmeric
extract is present at a concentration from about 2.5 weight percent
to about 25 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the turmeric
extract is present at a concentration from about 5 weight percent
to about 15 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the turmeric
extract is present at a concentration at least about 11 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the turmeric extract is present at
a concentration at least about 6 weight percent of the total dry
weight of active ingredients of the composition. In one embodiment,
the green tea extract is standardized to contain at least about 40%
polyphenols. In one embodiment, the at least one polyphenol is
polyphenol (-)-epigallocatechin gallate. In one embodiment, the
green tea extract is present at a concentration from about 2.5
weight percent to about 25 weight percent of the total dry weight
of active ingredients of the composition. In one embodiment, the
green tea extract is present at a concentration from about 5 weight
percent to about 15 weight percent of the total dry weight of
active ingredients of the composition. In one embodiment, the green
tea extract is present at a concentration at least about 11 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the green tea extract is present at
a concentration at least about 6 weight percent of the total dry
weight of active ingredients of the composition. In one embodiment,
the Gotu kola powder is present at a concentration from at least
about 5 weight percent to about 50 weight percent of the total dry
weight of active ingredients of the composition. In one embodiment,
the Gotu kola powder is present at a concentration from at least
about 10 weight percent to about 30 weight percent of the total dry
weight of active ingredients of the composition. In one embodiment,
the Gotu kola extract is present at a concentration at least about
12 weight percent of the total dry weight of active ingredients of
the composition. In one embodiment, the Ginko biloba leaf extract
is present at a concentration from at least about 5 weight percent
to about 50 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the Ginko biloba
leaf extract is present at a concentration from at least about 10
weight percent to about 30 weight percent of the total dry weight
of active ingredients of the composition. In one embodiment, the
Ginko biloba leaf extract is present at a concentration at least
about 12 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the Aloe vera
powder is present at a concentration from at least about 5 weight
percent to about 50 weight percent of the total dry weight of
active ingredients of the composition. In one embodiment, the Aloe
vera powder is present at a concentration from at least about 10
weight percent to about 30 weight percent of the total dry weight
of active ingredients of the composition. In one embodiment, the
Aloe vera powder is present at a concentration at least about 12
weight percent of the total dry weight of active ingredients of the
composition. In one embodiment, the N-acetyl cysteine is present at
a concentration of about 2 weight percent to about 20 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the N-acetyl cyteine is present at
a concentration of about 5 weight percent to about 15 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the N-acety cysteine is present at
a concentration of at least about 6 weight percent of the total dry
weight of active ingredients of the composition.
[0011] In one embodiment, the composition of the invention contains
at least two (2) of the active ingredients of the
antioxidant-promoting composition. In one embodiment, the
composition of the invention contains at least three (3) of the
active ingredients of the antioxidant-promoting composition. In one
embodiment, the composition of the invention contains at least four
(4) of the active ingredients of the antioxidant-promoting
composition. In one embodiment, the composition of the invention
contains at least five (5) of the active ingredients of the
antioxidant-promoting composition. In one embodiment, the
composition of the invention contains at least six (6) of the
active ingredients of the antioxidant-promoting composition. In one
embodiment, the composition of the invention contains at least
seven (7) of the active ingredients of the antioxidant-promoting
composition. In one embodiment, the composition of the invention
contains at least eight (8) of the active ingredients of the
antioxidant-promoting composition. In one embodiment, the
composition of the invention contains at least nine (9) of the
active ingredients of the antioxidant-promoting composition.
[0012] In another embodiment, the composition of the invention is
an antioxidant-promoting composition comprising active ingredients
comprising Bacopa monniera extract; milk thistle extract,
ashwagandha powder, green tea extract, and turmeric extract,
wherein the composition increases the enzyme activity level of at
least one antioxidant enzyme, e.g., superoxide dismutase; catalase;
and glutathione peroxidase and decreases the plasma concentration
level of thiobarbituric acid reactive chemical species, when
administered in an effective amount to a mammalian subject in need
thereof. In one embodiment, the Bacopa monniera extract is a
standardized extract prepared from the leaves of the Bacopa
monniera plant. In one embodiment, the standardized Bacopa monniera
extract contains at least 20% bacosides. In one embodiment, the
Bacopa monniera extract is present at a concentration of about 5
weight percent to about 50 weight percent of the total dry weight
of active ingredients of the composition. In one embodiment, the
Bacopa monniera extract is present at a concentration of about 10
weight percent to about 30 weight percent of the total dry weight
of active ingredients of the composition. In one embodiment, the
Bacopa monniera extract is present at a concentration at least
about 22 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the Bacopa
monniera extract is present at a concentration at least about 12
weight percent of the total dry weight of active ingredients of the
composition. In one embodiment, the milk thistle extract is
standardized to contain at least about 70 weight percent silymarin.
In one embodiment, the milk thistle extract is present at a
concentration from about 5 weight percent to about 60 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the milk thistle extract is present
at a concentration from about 10 weight percent to about 50 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the milk thistle extract is present
at a concentration at least about 33 weight percent of the total
dry weight of active ingredients of the composition. In one
embodiment, the milk thistle extract is present at a concentration
at least about 22 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the ashwagandha
powder is present at a concentration from about 5 weight percent to
about 50 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the ashwagandha
powder is present at a concentration from about 10 weight percent
to about 30 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment, the ashwagandha
powder is present at a concentration at least about 22 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the ashwagandha powder is present
at a concentration at least about 12 weight percent of the total
dry weight of active ingredients of the composition. In one
embodiment, the turmeric extract is standardized to contain at
least about 95 weight percent curcumin. In one embodiment, the
turmeric extract is present at a concentration from about 2.5
weight percent to about 25 weight percent of the total dry weight
of active ingredients of the composition. In one embodiment, the
turmeric extract is present at a concentration from about 5 weight
percent to about 15 weight percent of the total dry weight of
active ingredients of the composition. In one embodiment, the
turmeric extract is present at a concentration at least about 11
weight percent of the total dry weight of active ingredients of the
composition. In one embodiment, the turmeric extract is present at
a concentration at least about 6 weight percent of the total dry
weight of active ingredients of the composition. In one embodiment,
the green tea extract is standardized to contain at least about 40%
polyphenols. In one embodiment, at least one polyphenol is
polyphenol (-)-epigallocatechin gallate. In one embodiment, the
green tea extract is present at a concentration from about 2.5
weight percent to about 25 weight percent of the total dry weight
of active ingredients of the composition. In one embodiment, the
green tea extract is present at a concentration from about 5 weight
percent to about 15 weight percent of the total dry weight of
active ingredients of the composition. In one embodiment, the green
tea extract is present at a concentration at least about 11 weight
percent of the total dry weight of active ingredients of the
composition. In one embodiment, the green tea extract is present at
a concentration at least about 6 weight percent of the total dry
weight of active ingredients of the composition. In one embodiment,
the composition further comprises the active ingredients Gotu kola
powder, Ginko biloba leaf extract and Aloe vera powder. In one
embodiment, the Gotu kola powder is present at a concentration from
at least about 5 weight percent to about 50 weight percent of the
total dry weight of active ingredients of the composition. In one
embodiment, the Gotu kola powder is present at a concentration from
at least about 10 weight percent to about 30 weight percent of the
total dry weight of active ingredients of the composition. In one
embodiment, the Gotu kola powder extract is present at a
concentration at least about 12 weight percent of the total dry
weight of active ingredients of the composition. In one embodiment,
the Ginko biloba leaf extract is present at a concentration from at
least about 5 weight percent to about 50 weight percent of the
total dry weight of active ingredients of the composition. In one
embodiment, the Ginko biloba leaf extract is present at a
concentration from at least about 10 weight percent to about 30
weight percent of the total dry weight of active ingredients of the
composition. In one embodiment, the Ginko biloba leaf extract is
present at a concentration at least about 12 weight percent of the
total dry weight of active ingredients of the composition. In one
embodiment, the Aloe vera powder is present at a concentration from
at least about 5 weight percent to about 50 weight percent of the
total dry weight of active ingredients of the composition. In one
embodiment, the Aloe vera powder is present at a concentration from
at least about 10 weight percent to about 30 weight percent of the
total dry weight of active ingredients of the composition. In one
embodiment, the Aloe vera powder is present at a concentration at
least about 12 weight percent of the total dry weight of active
ingredients of the composition.
[0013] In some embodiments, the compositions of the present
invention further comprise at least one excipient, e.g., calcium
carbonate; croscarmellose sodium; dicalcium phosphate; magnesium
stearate; microcrystalline cellulose; modified cellulose; silica;
and stearic acid.
[0014] In another embodiment the invention provides an
antioxidant-promoting composition comprising: (a) active
ingredients comprising at least about 150 milligrams Bacopa
monniera, extract 45 percent bacosides; at least about 225
milligrams milk thistle extract, between about 70 percent and about
80 percent silymarin; at least about 150 mg ashwagandha powder; at
least about 75 milligrams green tea extract; 98 percent
polyphenols; 45 percent (-)-epigallocatechin gallate; at least
about 75 milligrams turmeric extract; 95 percent curcumin; and (b)
other ingredients comprising calcium carbonate; croscarmellose
sodium; dicalcium phosphate; magnesium stearate; microcrystalline
cellulose; modified cellulose; silica; and stearic acid; wherein
the composition increases the enzyme activity level of at least one
antioxidant enzyme selected from the group consisting of:
superoxide dismutase; catalase; and glutathione peroxidase and
decreases the plasma concentration level of thiobarbituric acid
reactive chemical species, when administered in an effective amount
to a mammalian subject in need thereof. In one embodiment, the
composition is formulated as an oral dosage form. In one
embodiment, the oral dosage form is selected from the group
consisting of: a tablet; capsule; and caplet.
[0015] In another aspect the invention provides a method of
increasing the antioxidant activity level of a mammalian subject in
need thereof, by increasing the level of enzyme activity of at
least one enzyme, e.g., superoxide dismutase; catalase; and
glutathione peroxidase, by administering to the subject an
effective amount of an antioxidant-promoting composition of the
invention, wherein the increased enzyme activity decreases the
tissue damage caused by pathological free radicals. In one
embodiment, the tissue damage caused by pathological free radicals
occurs in a mammalian subject with a disease or condition selected
from the group which includes, e.g., inflammation; infection;
atherosclerosis; hypertension; cancer; radiation injury;
neurological disease; neurodegenerative disease;
ischemia/reperfusion injury; aging; wound healing; glutathione
deficiency; acquired immunodeficiency syndrome; sickle cell anemia;
and diabetes mellitus. In one embodiment of the method, the
antioxidant-promoting composition is administered as an oral
dietary supplement.
[0016] In one embodiment, the invention provides a method of
reducing the plasma level of c-reactive protein in a mammalian
subject, by administering to the subject an effective amount of an
antioxidant-promoting composition of the invention. In one
embodiment of the method, the antioxidant-promoting composition is
administered as an oral dietary supplement. In one embodiment of
the method, the plasma level of c-reactive protein decreases by at
least 10% or more in a 30 day period. In one embodiment of the
method, the plasma level of c-reactive protein decreases by at
least 20% or more in a 30 day period.
[0017] In one embodiment, the invention provides a method of
reducing the blood pressure in a mammalian subject, by
administering to the subject an effective amount of an
antioxidant-promoting composition of the invention. In one
embodiment of the method, the antioxidant-promoting composition is
administered as an oral dietary supplement.
[0018] The compositions of the present invention are useful to
promote healthful benefits as follows: relaxation; bone marrow and
women's health (e.g., stabilizes fetus and regenerates hormones);
mental function (e.g., memory and concentration); sexual function;
diuretic; anti-inflammatory; anti-mutagenic agent; anti-cancer
agent; cholagogueue; depurative; fumitory; hemostatic agent;
hepatoprotective agent; lactagogue; stomachic; tonic; vulnerary;
tissue healing (e.g.; wounds; skin; other connective tissues; lymph
tissue; blood vessels; and mucous membranes); remove free radicals
in the peripheral and/or cerebral vascular systems; and inhibit
lipid peroxidation; improve cerebral blood circulation and to
protect the nerves against damaging free radicals; protective
against cell damage caused by chemotherapy and radiation therapy;
to enhance immune function; and to protect against toxins (e.g.,
acetametaphen and other drugs; mercury; lead).
[0019] The compositions of the present invention are useful to
prevent or treat the following disorders and diseases: memory loss;
Parkinson's disease; aging; toxin-induced hepatotoxicity;
inflammation; liver cirrhosis; chronic hepatitis; and diabetes due
to cirrhosis; indigestion; fatigue; stress; cough; infertility;
tissue inflammation; cancer; anxiety disorders; panic attacks;
rheumatism; pain; manic depression; alcoholic paranoia;
schizophrenia; fever; insomnia; infertility; aging; skin
inflammations and disorders; alcoholism; anemia; carbuncles;
convalescence; emaciation; HIV; AIDS; immune system problems;
lumbago; multiple sclerosis; muscle energy loss; paralysis; swollen
glands; ulcers; breathing difficulties; inflammation; psoriasis;
cancer (e.g.; prostate cancer, lung cancer and breast cancer);
pain; cardiovascular disease (e.g.; arteriosclerosis and
atherosclerosis); ischemia/reperfusion injury; anxiety; attention
deficit disorder; leprosy; arthritis (e.g., psoriatic arthritis;
anklylosing spondvlitis; and rheumatoid arthritis); hemorrhoids;
tuberculosis; high blood pressure; congestive heart failure; venous
insufficiency (pooling of blood in the veins; usually in the legs);
sore throat; hepatitis; syphilis; stomach ulcers; epilepsy;
diarrhea; asthma; burns; piles; sunburn; wrinkles; headache; insect
bites; cuts; ulcers; sores; herpes; jaundice; bursitis; canker
sores; sore gums; poison ivy; gastritis; high cholesterol; heart
disease; bacterial infection; viral infection; acne; aging; immune
disorders; dental caries; periodontitis; halitosis; dandruff;
cardiovascular disease (e.g., hypertension; thrombosis;
arteriosclerosis); migraine headaches, diabetes; elevated blood
glucose; diseases of the alimentary canal and respiratory system;
age-related physical and mental deterioration (e.g., Alzheimer's
Disease and age-related dementia); cardiovascular disease; cerebral
vascular insufficiency and impaired cerebral performance;
congestive symptoms of premenstrual syndrome; allergies;
age-related vision loss; depression; Raynaud's disease; peripheral
vascular disease; intermittent claudication; vertigo; equilibrium
disorder; prevention of altitude sickness; tinnitus (ringing in the
ear); liver fibrosis; macular degeneration; asthma; graft
rejection; and immune disorders that induce toxic shock;
bronchoulmonary disease as cystic fibrosis; chronic bronchitis;
gastritis; heart attack; angina pectoris; chronic obstructive
pulmonary disease; kidney damage during coronary angiography;
Unverricht-Lundborg disease; pseudoporphyria; pneumonia; and
paracetamol hepatotoxicity
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a graph illustrating effect of Protandim I dietary
supplementation on percent weight gain of mice after 23 days.
[0021] FIG. 2 shows graphs illustrating effect of Protandim I
dietary supplement on murine red blood cell SOD concentration (RBC
SOD). Panel A shows a graph of RBC SOD concentration (U/ml)
observed in mice fed 1.times., 3.times. and 10.times. dosage of
Protandim I dietary supplement for 23 days. Panel B shows a graph
of the percent increase in RBC SOD after 23 days on 1.times.,
3.times. and 10.times. dosage of Protandim I dietary supplement. An
asterisk indicated statistical significance at p<0.02 level.
[0022] FIG. 3 shows graphs effect of Protandim I dietary supplement
on murine liver SOD concentration. Panel A shows a graph of liver
SOD concentration (U/mg protein) observed in mice fed for 23 days
on 1.times., 3.times. and 10.times. dosage of Protandim I dietary
supplement. Panel B shows a graph of the percent increase in liver
SOD after 23 days on 1.times., 3.times. and 10.times. dosage of
Protandim I dietary supplement. An asterisk indicates statistical
significance at p<0.02 level. A double asterisk indicates
statistical significance at p<0.0001 level.
[0023] FIG. 4 shows graphs illustrating effect of Protandim I
dietary supplement on murine brain SOD concentration. Panel A shows
a graph of brain SOD concentration (U/mg protein) observed in mice
fed 1.times., 3.times. and 10.times. dosage of Protandim I dietary
supplement for 23 days. Panel B shows a graph of the percent change
in brain SOD after 23 days on 1.times., 3.times. and 10.times.
dosage of Protandim I dietary supplement. An asterisk indicates
statistical significance at p<0.02 level.
[0024] FIG. 5 shows graphs illustrating effect of Protandim I
dietary supplement on murine red blood cell catalase concentration
(RBC CAT). Panel A shows a graph of RBC CAT concentration (U/ml)
observed in mice fed 1.times., 3.times. and 10.times. dosage of
Protandim I dietary supplement for 23 days. Panel B shows a graph
of the percent increase in RBC CAT after 23 days on 1.times.,
3.times. and 10.times. dosage of Protandim I dietary
supplement.
[0025] FIG. 6 is a graph illustrating effect of Protandim I dietary
supplement on murine liver CAT concentration (U/mg protein)
observed in mice fed 1.times., 3.times. and 10.times. dosage of
Protandim I dietary supplement for 23 days.
[0026] FIG. 7 shows graphs effect of Protandim I dietary supplement
on murine red blood cell glutathione peroxidase concentration (RBC
GPX). Panel A shows a graph of RBC GPX concentration (U/ml)
observed in mice fed 1.times., 3.times. and 10.times. dosage of
Protandim I dietary supplement for 23 days. Panel B shows a graph
of the percent change in RBC GPX (U/g Hb) after 23 days on
1.times., 3.times. and 10.times. dosage of Protandim I dietary
supplement.
[0027] FIG. 8 shows graphs illustrating effect of Protandim I
dietary supplement on murine liver GPX concentration. Panel A shows
a graph of liver GPX concentration (U/mg protein) observed in mice
fed 1.times., 3.times. and 10.times. dosage of Protandim I dietary
supplement for 23 days. Panel B shows a graph of the percent change
in liver GPX after 23 days on 1.times., 3.times. and 10.times.
dosage of Protandim I dietary supplement. An asterisk indicated
statistical significance at p<0.004 level.
[0028] FIG. 9 shows graphs illustrating effect of Protandim I
dietary supplement on murine brain GPX concentration. Panel A shows
a graph of brain GPX concentration (U/mg protein) observed in mice
fed 1.times., 3.times. and 10.times. dosage of Protandim I dietary
supplement for 23 days. Panel B shows a graph of the percent change
in brain GPX after 23 days on various diets of a composition of the
present invention. An asterisk indicates statistical significance
at p<0.03 level.
[0029] FIG. 10 shows graphs illustrating effect of Protandim I
dietary supplement on murine plasma lipid peroxidation products
measured as TBARS. Panel A shows a graph of plasma TBARS
concentration (.mu.M) observed in mice fed 1.times., 3.times. and
10.times. dosage of Protandim I dietary supplement for 23 days.
Panel B shows a graph of the percent change in plasma TBARS after
23 days on 1.times., 3.times. and 10.times. dosage of Protandim I
dietary supplement. A single asterisk indicates statistical
significance at p<0.004 level. A double asterisk indicates
statistical significance at the p<0.0004 level.
[0030] FIG. 11 shows graphs illustrating the effect of diets of a
composition of the present invention on murine liver lipid
peroxidation products measured as TBARS. Panel A shows a graph of
liver TBARS concentration (nmol/mg protein) observed in mice fed
1.times., 3.times. and 10.times. dosage of Protandim I dietary
supplement for 23 days. Panel B shows a graph of the percent change
in liver TBARS after 23 days on 1.times., 3.times. and 10.times.
dosage of Protandim I dietary supplement. A single asterisk
indicates statistical significance at p=0.001 level. A double
asterisk indicates statistical significance at the p<0.00001
level.
[0031] FIG. 12 shows graphs illustrating effect of Protandim I
dietary supplement on murine brain lipid peroxidation products
measured as TBARS. Panel A shows a graph of brain TBARS
concentration (nmol/mg protein) observed in mice fed 1.times.,
3.times. and 10.times. dosage of Protandim I dietary supplement for
23 days. Panel B shows a graph of the percent change in brain TBARS
after 23 days 1.times., 3.times. and 10.times. dosage of Protandim
I dietary supplement. A single asterisk indicates statistical
significance at p<0.004 level. A double asterisk indicates
statistical significance at the p<0.0001 level.
[0032] FIG. 13 shows graphs illustrating effect of Protandim I
dietary supplement on murine SOD, CAT and GPX in various tissues.
Panel A shows a graph of the percent change in SOD observed in RBC,
liver and brain of mice fed 1.times., 3.times. and 10.times. dosage
of Protandim I dietary supplement for 23 days. Panel B shows a
graph of the percent change in CAT observed in RBC and liver of
mice fed 1.times., 3.times. and 10.times. dosage of Protandim I
dietary supplement for 23 days. Panel C shows a graph of the
percent change in GPX observed in RBC, liver and brain of mice fed
1.times., 3.times. and 10.times. dosage of Protandim I dietary
supplement for 23 days. A single asterisk indicates statistical
significance at relative to the control diet.
[0033] FIG. 14 is a graph illustrating the effect of Protandim I
dietary supplement on murine lipid peroxidation in various tissues.
The graph shows the percent change in lipid peroxidation measured
as TBARS in RBC, liver and brain of mice fed 1.times., 3.times. and
10.times. dosage of Protandim I dietary supplement for 23 days. A
single asterisk indicates statistical significance at relative to
the control diet.
[0034] FIG. 15 is a graph showing response of human subjects to
dietary supplementation with the herbal composition Protandim II.
Panel A shows plasma TBARS level in human subjects prior to
supplementation with Protandim II at 675 mg/day (closed circles),
after 30 days of supplementation (gray squares), and after 120 days
(open circles). The levels of plasma TBARS dropped an average of
51% (p<0.002) after 30 days of Protandim treatments (gray
squares) the age-related increase in TBARS virtually disappeared.
Panel B shows plasma TBARS level in normal subjects before
supplementation with Protandim II showed a strong age-dependent
increase in TBARS (circles). The levels of plasma TBARS dropped on
average 51% (p<0.002) after 30 days of Protandim II (squares)
supplementation, and the age-related increase in TBARS virtually
disappeared.
[0035] FIG. 16 is a graph illustrating the effect of Protandim II
dietary supplementation on human SOD in RBCs.
[0036] FIG. 17 is a graph illustrating the effect of Protandim II
dietary supplementation on human CAT in RBCs.
[0037] FIG. 18 is a graph illustrating the effect of Protandim II
dietary supplementation on human CRP protein level.
[0038] FIG. 19 is a graph illustrating the effect of half-dose
Protandim II on plasma TBARS in humans subjects. The half-dose
Protandim II was 338 mg/day dietary supplement.
[0039] FIG. 20 is a graph comparing the effect of Protandim II
dosage on plasma TBARS in human subjects. Average plasma TBARS
concentrations are shown after 30 days supplementation of Protandim
II at full-dose (675 mg/day; n=11) and half-dose (338 mg/day; n=4).
The difference was statistically significant at p<0.03 as
determined by one-tailed paired t-test.
[0040] FIG. 21 shows graphs detailing the blood pressure
measurements of human subjects administered an herbal composition
of the invention. Panel A shows the blood pressure measurements for
Subject #2 in Example 2. Panel B shows the blood pressure
measurements for Subject #4 in Example 2. The arrow indicates the
point in time at which the individual began treatment with an
herbal composition of the invention. In both individuals, diastolic
blood pressure dropped significantly over a period of about 21
days, remaining constant thereafter.
DETAILED DESCRIPTION OF THE INVENTION
[0041] There remains a need to formulate improved compositions and
supplements that increase the SOD, CAT, or GPX levels in a mammal,
that may be more effective and have fewer side-effects and lowered
toxicity. The present invention provides compositions that can be
administered to a mammalian subject, and will safely induce in the
subject, increased antioxidant potential in the subject by
increasing the activity of at least one antioxidant enzyme which
include, e.g., SOD, CAT, and GPX, and thereby decreasing the tissue
level of pathologic free radical species. The composition provides
for an overall net decrease in oxidative stress experienced by the
mammal, with minimal undesirable side-effects. Advantageously, the
composition itself provides fewer side effects than may be
associated with each of the individual active agents in the
composition.
[0042] Accordingly, the various aspects of the present invention
relate to therapeutic or prophylactic uses of certain particular
compositions (e.g., dietary supplement compositions) in order to
prevent or treat a disease or an injury induced by pathological
free and reactive radical reactions. The various aspects of the
present invention further relate to therapeutic or prophylactic
uses of certain particular compositions in order to prevent or
treat a disease or an injury associated with decreased SOD, CAT,
and/or GPX enzyme activity, or that would benefit from increased
SOD, CAT, and/or GPX enzyme activity, such as inflammation and
oxidative stress.
[0043] Various particular embodiments that illustrate these aspects
follow.
[0044] It is to be appreciated that the various modes of treatment
or prevention of medical conditions as described are intended to
mean "substantial", which includes total but also less than total
treatment or prevention, and wherein some biologically or medically
relevant result is achieved. A "subject," as used herein, is
preferably a mammal, such as a human, but can also be an animal,
e.g., domestic animals (e.g., dogs, cats and the like), farm
animals (e.g., cows, sheep, pigs, horses and the like) and
laboratory animals (e.g., rats, mice, guinea pigs and the like). An
"effective amount" of an antioxidant inducing composition, as used
herein, is a quantity of the composition provided by a particular
route of administration and at a particular dosing regimen, that is
sufficient to achieve a desired therapeutic and/or prophylactic
effect. For example, an amount that results in the prevention of or
a decrease in the symptoms associated with a disease that is being
treated. The amount of the composition administered to the subject
will depend on the type and severity of the disease, the
amenability of the disorder to respond to antioxidants, and on the
characteristics of the individual and their metabolic ability to
respond to the compositions to produce in vivo, the antioxidants,
such factors including general health, age, sex, body weight and
tolerance to the active agents in the compositions. The skilled
artisan will be able to determine appropriate dosages depending on
these and other factors. Typically, an effective amount of the
compositions of the present invention, sufficient for achieving a
therapeutic or prophylactic effect, range from about 0.000001 mg
per kilogram body weight per day to about 10,000 mg per kilogram
body weight per day. Preferably, the dosage ranges are from about
0.0001 mg per kilogram body weight per day to about 100 mg per
kilogram body weight per day. The compositions of the present
invention can also be administered in combination with each other,
or with one or more additional therapeutic or prophylactic
compounds.
[0045] It is advantageous to formulate oral compositions in dosage
unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units
suited as unitary dosages for the subject to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on the unique characteristics of the dietary supplement and the
particular therapeutic effect to be achieved, and the limitations
inherent in the art of compounding such an active compound for the
treatment of individuals. The pharmaceutical compositions can be
included in a container, pack, or dispenser together with
instructions for administration. Typically, an oral dose is taken
two-times to four-times daily, until symptom relief is apparent.
The compositions of the present invention can also be administered
in combination with each other, or with one or more additional
therapeutic compounds.
[0046] It is to be appreciated that certain aspects, modes,
embodiments, variations and features of the invention are described
below in various levels of detail in order to provide a substantial
understanding of the present invention. In general, such disclosure
provides compositions beneficial to the health of a subject. In one
embodiment, the compositions of the invention is a dietary
supplement composition. In another embodiment, the compositions of
the invention are combined with other dietary supplement
compositions. In yet other embodiments, the composition of the
invention is a pharmaceutical formulation.
Radicals, Oxidative Stress and Cellular Defense
[0047] Over the past few decades, free and bound reactive radicals,
highly reactive and thereby destructive molecules, have come to be
appreciated increasingly for their importance to human health and
disease. Many common and life-threatening human diseases, including
atherosclerosis, cancer, and aging, have radical-based pathological
reactions as an underlying mechanism of injury.
[0048] A radical, or a free radical, is generally understood as a
molecule with one or more unpaired electrons in its outer orbital
shell. Many molecular species with bound radicals are monoxides or
other oxygen containing compounds, generally referred to as
reactive oxygen species (ROS). These highly unstable molecules tend
to react rapidly with adjacent molecules, donating, abstracting, or
even sharing their outer orbital electron(s). This reaction not
only changes the adjacent, target molecule, sometimes in profound
and beneficial ways, but it can also damage it, or alternatively
the unpaired electron can be passed along to the target, i.e., as
in a free radical, generating a second unwanted ROS, which can then
go on to react positively or detrimentally with a new target. In
fact, much of the high reactivity of ROS is due to their generation
of such molecular chain reactions, effectively amplifying their
effects many fold. Antioxidants afford protection because they can
scavenge ROS and free radicals before they cause damage to the
various biological molecules, or prevent oxidative damage from
spreading, e.g., by interrupting the radical chain reaction of
lipid peroxidation. The reactivity of radicals in the body, and the
burden on the body that results in eventual pathological
conditions, is known as oxidative stress.
[0049] For example, oxidative stress in the circulatory system is
seen as atherosclerosis and by plaque deposition on the vessel
walls. Plaques cause inflammatory responses, which increase radical
formation by recruited immune cells, which worsens the cycle. It
has been observed that the prime targets of both free radicals and
ROS are the polyunsaturated fats in the membrane lipids of cells.
The oxidative deterioration of polyunsaturated fats is known as
lipid peroxidation. Lipid peroxidation severely impairs membrane
function, which is believed to lead to the disorganization of cell
structure and function. Products of lipid peroxidation such as
malondialdehyde, a known mutagen reactive with proteins and amino
acids, are a good measure of the amount of oxidative stress on the
body. Lipofuscin, another byproduct of lipid peroxidation,
accumulates in the body with age and it is believed that cytosolic
buildup of this byproduct compromises brain function. What begins
as localized pathological radical reactions thus result in
oxidative stress that can and will eventually impact distal organ
systems.
[0050] Vitamins such as vitamin C and vitamin E, both of which are
found in foods and available as supplements, help the body reduce
effects of oxidative stress. A more powerful combatant against the
free radicals and ROS, however, is the body's own self defense
system of naturally produced chemicals called antioxidants. These
antioxidants act to terminate the propagation of free and bound
radicals on ROS either by giving an electron to the free radical or
ROS or by hindering their formation.
[0051] The body's antioxidant defense system includes three
important natural antioxidants: superoxide dismutase (SOD),
catalase (CAT), and glutathione peroxidase (GPX). Studies conducted
have indicated that these antioxidants can work synergistically as
the reactions they catalyze are metabolically sequential, beginning
first with SOD followed by the actions of CAT and GPX.
[0052] SODs are a class of enzymes that catalyze reactions similar
to the one below:
O2-+O2-+2H+.fwdarw.H2O2+O2
[0053] The role of SOD in the defense system is to remove
superoxide radicals, which are a type of ROS. SOD is found in the
body, primarily in three forms: (1) in the cytoplasm as Cu--Zn SOD;
(2) in the mitochondrion as Mn-SOD; (3) and in an extracellular
environment as CuSOD. Accordingly, the body is dependent upon the
presence of zinc, copper, and manganese for the manufacture of SOD.
During the removal of superoxide radicals by SOD, both hydroxyl and
oxygen radicals are produced, which are catalyzed by CAT and GPX
respectively.
[0054] Catalase (CAT) is present in the peroxisomes of nearly all
aerobic cells, and serves to protect the cell from the toxic
effects of hydrogen peroxide by catalyzing its decomposition into
molecular oxygen and water without the production of free
radicals.
[0055] The overall reaction is as follows:
2H2O2.fwdarw.2H2O+O2
[0056] The protein exists as a dumbbell-shaped tetramer of four
identical subunits (220,000 to 350,000 kDa). Each monomer contains
a heme prosthetic group at the catalytic center. CAT monomers from
certain species (e.g., cow) also contain one tightly bound NADP per
subunit. This NADP may serve to protect the enzyme from oxidation
by its H2O2 substrate. The body's production of CAT is dependent
upon the availability of iron.
[0057] Glutathione peroxidases (GPXs) are a large class of diverse
enzymes which catalyze the reduction of hydrogen peroxide (H2O2),
organic hydroperoxides and lipid hydroperoxides to the
corresponding alcohol using glutathione (GSH;
glutamylcysteinylglycine) as the electron donor (Ursini et al.,
1995). The GPX enzymes catalyze the reduction of H2O2 to water and
organic peroxides (R--O--O--H) to the corresponding stable alcohols
(R--O--H) using glutathione (GSH) as a source of reducing
equivalents:
2GSH.fwdarw.ROH+GSSG+H2O
[0058] GPXs are important in helping to protect cells against
oxidative damage, particularly lipid peroxidation. (Flohe et al.,
1976). In mammals, the cytosolic (c) GPX family as well as a family
of phospholipid hydroperoxide (PH) GPXs possess a selenocysteine
residue at their active site. With the exception of
phospholipid-hydroperoxide GPX, a monomer, all of the GPX enzymes
are comprised of four identical subunits (monomer Mr 22-23 kDa).
Each subunit contains a molecule of selenocysteine in the enzyme
active site. The selenocysteine is thought to participate directly
in electron donation to the peroxide substrate and become oxidized
in the process. The enzyme then uses glutathione as an electron
donor to regenerate the reduced form of the selenocysteine (Ursini,
1995). The GPX enzymes accept a wide variety of organic peroxides
as substrates. The body's ability to produce GPX is in part
dependent upon the adequate supply of selenium, and the supply of
glutathione, a tripeptide that the body produces from the amino
acids cysteine, glutamic acid, and glycine. Mammals contain plasma
(p) GPXs, which are nonselenium containing enzymes in which
cysteine replaces selenocysteine at their active site (Ursini et
al., 1995). However, with the exception of phospholipid
hydroperoxide GPX and perhaps pl.cndot.GPX, the enzymes exhibit a
strong preference for glutathione as a source of reducing
equivalents.
[0059] Despite the presence of SOD, CAT, and GPX, the body's
antioxidant defense system is constantly subject to oxidative
stress, and its ability to produce SOD, CAT, and GPX is compromised
by the aging process and can further be impaired by inflammation,
microbial or viral infections, the progression of cancer and
neurological disorders, and other pathological conditions that
produce, are caused by, or are exacerbated by oxidative stress.
Herb-Containing Compositions of the Invention
[0060] One object of the present invention is to provide new and
useful compositions that alleviate oxidative stress in a subject.
Another object of the present invention is to provide compositions
that assist the body's natural antioxidant defense system(s) to
alleviate the harm associated with oxidative stress. A further
object of the present invention is to provide new compositions that
upregulates the levels of SOD, CAT, and GPX, lowers the
concentrations of free radicals and ROS, and decreases the rate of
lipid peroxidation. Also, an object of the present invention is to
provide dietary supplement compositions that alleviate oxidative
stress as well as provide the body with other healthful benefits.
Yet another object of the present invention is to formulate a
composition in the form of a tonic or capsule, with the beneficial
effects of alleviating oxidative stress that is of a sufficient
concentration that may be easily implemented as part of a daily
supplement regime. As noted above, the compositions of the
invention provide for an overall net decrease in oxidative stress
experienced by the mammal, with minimal undesirable side-effects.
Advantageously, the composition itself provides fewer side effects
than may be associated with each of the individual active agents in
the composition.
[0061] These and other objects of the present invention will become
more readily appreciated and understood from a consideration of the
following detailed description of the present invention.
[0062] The present invention provides compositions derived from
plant sources that induce the body's production of the antioxidants
SOD, CAT, and GPX, and to a method of alleviating oxidative stress
through use of these compositions. The compositions provide for
efficient upregulation of these antioxidant enzymes enzymes, but
advantageously the composition has lowered side-effects, i.e.,
reduced toxicity in the combination relative to the toxicity of
each active agent when administered alone.
[0063] Observations related to lipid peroxidation are a good
measure as to the amount of oxidative stress in a subject. Ideally,
natural dietary supplements that induce production of SOD, CAT and
GPX should also have the effect of reducing the concentration of
free radicals and ROS in the body and result in decreased rates of
lipid peroxidation. However, it has been observed that some
formulations of dietary supplements that induce antioxidant
production may contemporaneously cause an increase in the
concentration of ROS leading to increased rates of lipid
peroxidation. In other words, while some supplements may achieve an
increase in the body's production of SOD, CAT, and GPX, it leaves
the body with an overall net increase in oxidative stress. The
present compositions do not add to the oxidative stress of the
body.
[0064] Accordingly, choosing an active agent based simply on its
ability to cause the induction of antioxidant production, per se,
may not be an adequate selection criterion to achieve the overall
desired goal of alleviating oxidative stress. Furthermore, plant
extracts, which are typically the ingredients of natural dietary
supplements, generally have their respective undesirable
side-effects, that also need to be properly balanced in an overall
formulation that accomplishes a dilution of individual undesirable
effects. For example, research has indicated that an ethanol
extract of Bacopa monniera produces toxicity in the brine shrimp
lethality assay at about 300 mg/L (D'Souza et al. Phytotherapy Res.
2002, vol 16, 197-8) and enhances thyroid function, which may be an
undesirable side effect (Kar et al. J. Ethnopharmacol. 2002, vol
81, 281-5).
[0065] The foregoing problems, among others, have been resolved by
the present invention. Specifically, the present invention provides
compositions containing appropriate mixtures of plant extracts are
to safely induce antioxidant production to achieve an overall net
decrease in oxidative stress, while at the same time diluting or
reducing the undesirable side-effect and toxicity profiles
associated with the individual extracts that make up the
composition or any pharmaceutically acceptable formulation
thereof.
[0066] The mixture contemplated by the present invention is
comprised of the following nine plant derived active agents, which
are listed in the following table along with the desired amounts of
each agent. In one aspect, an antioxidant inducing agent further
comprises any of the following nine plant derived active agents,
which are incorporated into the antioxidant-inducing preparation in
currently preferred concentrations (wt/wt) ranging from about 0.001
mg to about 1000 g, preferably about 0.1 mg to about 10 g, more
preferably about 1 mg to about 2 grams, and most preferably about
50 mg to about 500 mg.
TABLE-US-00001 TABLE 1 AMOUNT Per dosage ACTIVE AGENTS unit
POSSIBLE UNDESIRED EFFECTS Bacopa monniera extract 0.001 mg
Considered very safe, but shows cytotoxicity to 1000 g in cultured
cells (100 mg/kg) and anticancer activity (D'Souza et al.,
Phytotherapy Res. 2002, vol 16, 197-8); enhances thyroid function
(200 mg/kg) (Kar et al. J. Ethnopharmacol. 2002, vol 81, 281-5)
Gotu Kola powder 0.001 mg Sedative and antidepressive effects;
Possible to 1000 g hypotensive effect (PDR for Herbal Medicines
(First Edn). Medical Economics Co., 1998, 729-30) Ashwagandha
powder 0.001 mg Considered very safe, but shows to 1000 g
tranquilizing and hypotensive effects at 25 mg/kg (Mishra et al.
Altern. Med. Rev. 2000, vol 5, 334-346); (Malhotra et al., Indian
J. Physiol Pharmacol. 1965, vol 9, 127-136) Green tea 98% 0.001 mg
Stimulant (caffeine); possible gastric irritant Polyphenols 45%
EGCG to 1000 g (PDR for Herbal Medicines (First Edn). Medical
Economics Co., 1998, 710-1) Turmeric extract 95% 0.001 mg Possible
gastric irritant; possible antifertility to 1000 g effects at 125
mg/kg (PDR for Herbal Medicines (First Edn). Medical Economics Co.,
1998, 786-7) Milk Thistle extract 0.001 mg Considered very safe,
but causes increases 70-80% to 1000 g cell division in vitro (PDR
for Herbal Medicines (First Edn). Medical Economics Co., 1998,
1138-9); May be proinflammatory at high doses of 250 mg/kg (Johnson
et al. Planta Med. 2003, vol. 69, pp. 44-49) Aloe vera powder 0.001
mg Laxative effect; Gastrointestinal cramping to 1000 g (PDR for
Herbal Medicines (First Edn). Medical Economics Co., 1998, 630-3);
inhibits thyroid function at 125 mg/kg (Kar et al., J.
Ethnopharmacol. 2002, vol 81, 281-5) Ginko biloba leaf extract
0.001 mg Mild gastrointestinal irritant; hypersensitivity to 1000 g
reactions; may interfere with antithrombotic therapy at 240 mg/day
(PDR for Herbal Medicines (First Edn). Medical Economics Co., 1998,
871-3) N-Acetyl Cysteine 0.001 mg Considered Safe to 1000 g
TABLE-US-00002 TABLE 2 ACTIVE AGENTS DOSAGE/DAY Bacopa monniera
extract 10-4,000 mg Milk Thistle extract 70-80% 15-6,000 mg
Ashwagandha powder 10-4,000 mg Turmeric extract 95% 5-2,000 mg Gotu
Kola powder 10-4,000 mg Aloe vera powder 10-4,000 mg Green tea, 98%
Polyphenols 45% EGCG 5-2,000 mg Ginko biloba leaf extract 5-2,000
mg N-Acetyl Cysteine 50-5,000 mg
[0067] As shown in Table 1, many of the plant extracted active
agents listed have the possibility of undesirable side-effects at
high doses. However, by using a combination of some or all of these
nine active agents in the respective amounts as listed in Table 2
to formulate a plant based composition (e.g., an oral dietary
supplement), the desirable effects are additive and in certain
embodiments, even synergistic. The overall effect then, is to
upregulate at least one antioxidant enzyme, e.g., SOD, CAT, and
GPX, while decreasing the concentration of free radicals and ROS
and the rate of lipid peroxidation and other undesirable chemical
reactions that result in oxidative stress on the body. For example,
tissue level of thiobarbituric acid reactive chemical species
(e.g., plasma TBARS) are reduced after administration of a
composition of the invention to a subject. Furthermore, the
likelihood of observing the undesired side-effects listed for each
respective active agent is more remote as each extract is diluted
out by a factor of up to nine (9).
[0068] Further, it is expected that the above-described active
agents, in the amounts listed, will provide a combined remedy for
oxidative stress that may be processed, in one embodiment, into a
dosage unit for oral administration. This dosage unit is then
administered, as a tablet, capsule, gel cap, pellet (globule), or
in other carrier suitable for oral administration. Alternatively,
the composition could be made available as a powder to be mixed
with a suitable liquid, such as water, to form a tonic. In one
embodiment, the formulation of the herb-containing composition of
the invention is an oral dietary supplement. As such, an efficient,
proper, and effective balance of these active agents can be
formulated as to provide a composition that can be administered as
a suitable daily oral dietary supplement.
[0069] In one embodiment, the herb-containing composition of the
invention contains at least two (2) of the components (e.g.,
ingredients) summarized in Table 2. In some embodiments, the
herb-containing composition of the invention contains two (2),
three (3), four (4), five (5), six (6), seven (7), eight (8), or
nine (9) of the components summarized in Table 2. The
herb-containing composition of the invention can contain the
components summarized in Table 2 in any quantity, or combination,
suitable to give the desired oxidant preventative, therapeutic, or
alleviating effect.
Antioxidant Properties and Uses of the Compositions of the
Invention
[0070] The invention provides a method of preventing, alleviating
or treating oxidative stress in a subject. The active agents of the
invention are formulated into a composition that retains the
prophylactic and therapeutic antioxidant inducing properties of the
individual active agents, providing an additive or even synergistic
antioxidant inducing effect relative to the effect of each active
alone, while also decreasing the toxic side effect(s) to a subject,
of the individual active agents of the compositions. The
compositions of the invention are useful to eradicate free and
bound radical reactions presently taking place or it may be used as
prophylaxis against pathological free or bound radical reactions,
which may occur as a result of a possible oxidant promoting
incident (e.g., ischemic injury).
[0071] The present invention provides compositions for increasing
the levels of antioxidants, via alteration of the activity level of
SOD, CAT, and GPX enzymes in the body. The composition provides in
one embodiment, a mixture of herbal extracts of Bacopa monniera (B.
monniera or Bacopa), which contains a high percentage of the active
chemicals bacosides A & B. Ingestion of Bacopa induces SOD,
CAT, and GPX and provides the beneficial activities thereof, with
pronounced results in the brain. Studies have indicated that the
bacosides also increase protein and serotonin levels, while
decreasing norepinephrine concentration in the hippocampus,
hypothalamus, and cerebral cortex. Bacopa thus reduces the
neurodegeneration in the brain that is caused by oxidative stress
related to the accumulation of neurotoxic free radicals in the
brain. Accordingly, it may be used to alleviate symptoms of
neurodegenerative disorders, such as memory loss, Alzheimer's
disease, and Parkinson's disease, and even aging.
[0072] Bacopa monniera
[0073] Bacopa monniera (common names: water hyssop and Brahmi) is a
creeping perennial that thrives in warmer temperate climates. The
genus Bacopa includes over 100 species of aquatic herbs distributed
throughout the warmer regions of the world. The plant is a
profusely branched herb, rooting at the nodes and forming dense
mats. B. monniera extract (Bacopin.RTM.) is a standardized extract
prepared from the leaves of the B. monniera plant (Sabinsa
Corporation, Piscataway, N.J., USA). It is standardized for a
minimum of 20% bacosides A & B, the active ingredients
beneficial in the support of cognitive functions. Other extracts of
the B. monniera plant standardized for a greater minimum levels of
bacosides A & B (e.g., 30%, 40%, 50%, etc.) are useful in the
compositions of the present invention and can be prepared by
extraction techniques known in the art. The pharmacological effects
of B. monniera preparations/extracts also include antioxidant,
anti-inflammatory, cardiotonic and anticancer effects. Extract of
B. monniera is commercially available, e.g., Viable Herbal
Solutions (Morrisville, Pa., USA).
[0074] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, from about 10 mg to about
4,000 mg B. monniera extract is administered to a subject daily
using an herb-containing composition of the invention (See
generally, Table 2). In one embodiment of the method preventing,
alleviating or treating oxidative stress in a subject, from about
50 mg to about 3,000 mg B. monniera extract is administered to a
subject daily using an herb-containing composition of the
invention. In one embodiment of the method preventing, alleviating
or treating oxidative stress in a subject, from about 100 mg to
about 2,000 mg B. monniera extract is administered to a subject
daily using an herb-containing composition of the invention. In one
embodiment of the method preventing, alleviating or treating
oxidative stress in a subject, at least about 200 mg B. monniera
extract is administered to a subject daily using an herb-containing
composition of the invention. In one embodiment, the B. monniera
extract of the herb-containing composition is Bacopin.RTM.. In one
embodiment, the herb-containing composition of the invention
contains a B. monniera extract standardized for a at least about
20% bacosides A & B. In another embodiment, the herb-containing
composition of the invention contains a B. monniera extract
standardized for at least about 30% bacosides A & B. In another
embodiment, the herb-containing composition of the invention
contains a B. monniera extract standardized for at least about 40%
bacosides A & B. In another embodiment, the herb-containing
composition of the invention contains a B. monniera extract
standardized for at least about 50% bacosides A & B.
[0075] Milk Thistle
[0076] Milk thistle (botanical name; Silybum marianum; other common
names: Marian, Silybum, Silymarin) is a fine, tall plant, about the
size of the Cotton Thistle, with cut into root-leaves, waved and
spiny at the margin, of a deep, glossy green, with milk white
veins, and is found not uncommonly in hedgebanks and on waste
ground. Useful parts of the plant include, e.g., the whole herb,
root, leaves, seeds and hull. Milk thistle seeds contain a
bioflavonoid complex known as silymarin. Silymarin is an extract of
the seeds of the milk thistle plant. A standardized extract should
be 80% silymarin (the active ingredient). This constituent is
responsible for the medical benefits of the plant. Silymarin is
made up of three parts: silibinin, silidianin, and silicristin.
Silibinin is the most active and is largely responsible for the
benefits attributed to silymarin. As with other bioflavonoids,
silymarin is a powerful antioxidant. Milk thistle extract is useful
to protect or reverse damage to liver cells from toxins (e.g.,
alcohol, drugs, pesticides, poisons), to promote the regeneration
of liver cells, to prevent or treat liver disease (e.g., liver
cirrhosis, chronic hepatitis, and diabetes due to cirrhosis),
indigestion, and cancer. Silymarin's effect in preventing liver
destruction and enhancing liver function relates largely to its
ability to inhibit the factors that are responsible for hepatic
damage, i.e., free radicals and leukotrienes, coupled with an
ability to stimulate liver protein synthesis. Milk thistle (80%
silymarin) extract is commercially available, e.g., Stayleaner.com
(Las Vegas, Nev., USA).
[0077] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, from about 15 mg to about
6,000 mg milk thistle extract (70%-80%) is administered to a
subject daily using an herb-containing composition of the invention
(See generally, Table 2). In one embodiment of the method
preventing, alleviating or treating oxidative stress in a subject,
from about 50 mg to about 5,000 mg milk thistle extract (70%-80%)
is administered to a subject daily using an herb-containing
composition of the invention. In one embodiment of the method
preventing, alleviating or treating oxidative stress in a subject,
from about 100 mg to about 3,000 mg milk thistle extract (70%-80%)
is administered to a subject daily using an herb-containing
composition of the invention. In one embodiment of the method
preventing, alleviating or treating oxidative stress in a subject,
at least about 300 mg milk thistle extract (70%-80%) is
administered to a subject daily using an herb-containing
composition of the invention.
[0078] Ashwagandha
[0079] Ashwagandha (botanical names: Withania somnifera and
Physalis flexuosa; other common names: winter cherry, Ashgandh,
Achuvagandi, Amikkira-gadday, Amkulang-kalang, Amukkira-kilzhangu,
Amukran-kizhangu, Asagandha, Asana, Asgandh, Asundha, Asvagandhi,
Fatarfoda, Hirimaddina-gadday, Hirre-gadday, Penneroo-gadda,
Pevette, Sogade-beru, Indian ginseng) is an erect branched shrub
native to India, Pakistan and Sri Lanka. Ashwagandha preparations
are useful to promote relaxation, bone marrow and women's health
(e.g. stabilizes fetus and regenerates hormones), to enhance mental
function (e.g., memory and concentration), as an aphrodisiac, and
to treat fatigue, stress, cough, infertility, tissue inflammation,
cancer, infectious disease, anxiety disorders, panic attacks,
rheumatism, arthritis, pain, manic depression, alcoholic paranoia,
and schizophrenia, fever, insomnia, infertility, aging, skin
inflammations and disorders, alcoholism, Alzheimer's disease,
anemia, carbuncles, convalescence, emaciation, HIV support, AIDS,
immune system problems, lumbago, multiple sclerosis, muscle energy
loss, paralysis, skin afflictions, swollen glands, ulcers, as well
as breathing difficulties and as a diuretic. Ashwaganda powder is
commertically available, e.g., iHerb Inc. (Monrovia, Calif.,
USA).
[0080] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, from about 10 mg to about
4,000 mg Ashwagandha powder is administered to a subject daily
using an herb-containing composition of the invention (See
generally, Table 2). In one embodiment of the method preventing,
alleviating or treating oxidative stress in a subject, from about
50 mg to about 3,000 mg Ashwagandha powder is administered to a
subject daily using an herb-containing composition of the
invention. In one embodiment of the method preventing, alleviating
or treating oxidative stress in a subject, from about 100 mg to
about 2,000 mg Ashwagandha powder is administered to a subject
daily using an herb-containing composition of the invention. In one
embodiment of the method preventing, alleviating or treating
oxidative stress in a subject, at least about 200 mg Ashwagandha
powder is administered to a subject daily using an herb-containing
composition of the invention.
[0081] Turmeric
[0082] Turmeric extract 95% is prepared from the root or rhizome of
the Curcuma longa plant (common names: Curcuma, Turmeric, Ukon,
Goeratji, Kakoenji, Koenjet, Kondin, Kunir, Kunyit, Oendre, Rame,
Renet, Temu kuning, Temu kunyit, Tius. Curcumin). C. longa is a
perennial plant native to India. A compound called curcumin is a
potent extract of the root, and has been attributed a wide range of
therapeutic benefits. Turmeric extract is useful as an antioxidant,
anti-inflammatory, anti-mutagenic agent, anti-cancer agent,
cholagogueue, depurative, diuretic, fumitory, hemostatic agent,
hepatoprotective agent, lactagogue, stomachic, tonic, and
vulnerary. Turmeric preparations are useful to protect the liver
from toxins, to reduce platelet aggregation, to prevent or treat
inflammatory disease, inflammation, arthritis, psoriasis, cancer
(e.g., prostate cancer and breast cancer), pain, Alzheimer's
Disease, cardiovascular disease (e.g., arteriosclerosis and
atherosclerosis). Turmeric extract that is standardized to 95%
curcumin contains turmeric (with 95% curcumin). Turmeric extract
95% is commercially available, e.g., EZ-FITNESS (Northborough, M A,
USA).
[0083] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, from about 5 mg to about
2,000 mg Turmeric extract (95%) is administered to a subject daily
using an herb-containing composition of the invention (See
generally, Table 2). In one embodiment of the method preventing,
alleviating or treating oxidative stress in a subject, from about
10 mg to about 1,000 mg Turmeric extract (95%) is administered to a
subject daily using an herb-containing composition of the
invention. In one embodiment of the method preventing, alleviating
or treating oxidative stress in a subject, from about 50 mg to
about 500 mg Turmeric extract (95%) is administered to a subject
daily using an herb-containing composition of the invention. In one
embodiment of the method preventing, alleviating or treating
oxidative stress in a subject, at least about 100 mg Turmeric
extract (95%) is administered to a subject daily using an
herb-containing composition of the invention.
[0084] Gotu kola
[0085] Gotu kola (botanical names: Hydrocotyle asiatica, Centella
asiatica; other common names: Centella, March Pennywort, Indian
Pennywort, Hydrocotyle, Brahmi (Sanskrit), Luei Gong Gen (Chinese))
is a slender, creeping perennial plant that grows commonly in
swampy areas of India, Sri Lanka, Madagascar, South Africa and the
tropics. Gotu kola is distinct from the kola nut. Gotu kola powder
is prepared from the leaves and aerial parts of the plant and used
for medicinal purposes. Gotu kola powder is useful to promote
relaxation, to enhance mental function (e.g., memory and
concentration), to promote tissue healing (e.g., wounds, skin,
other connective tissues, lymph tissue, blood vessels, and mucous
membranes) and to treat fatigue, anxiety, attention deficit
disorder, insomnia, skin inflammations, leprosy, cancer, arthritis
(e.g., psoriatic arthritis, anklylosing spondvlitis, and rheumatoid
arthritis), hemorrhoids, tuberculosis, high blood pressure,
congestive heart failure, venous insufficiency (pooling of blood in
the veins, usually in the legs), sore throat, hepatitis, syphilis,
stomach ulcers, epilepsy, diarrhea, fever, and asthma and as a mild
diuretic. Gotu kola powder is commercially available, e.g.,
.RTM.Internatural (Twin Lakes, Wis., USA).
[0086] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, from about 10 mg to about
4,000 mg Gotu kola powder is administered to a subject daily using
an herb-containing composition of the invention (See generally,
Table 2). In one embodiment of the method preventing, alleviating
or treating oxidative stress in a subject, from about 25 mg to
about 2,000 mg Gotu kola powder is administered to a subject daily
using an herb-containing composition of the invention. In one
embodiment of the method preventing, alleviating or treating
oxidative stress in a subject, from about 50 mg to about 1,000 mg
Gotu kola powder is administered to a subject daily using an
herb-containing composition of the invention. In one embodiment of
the method preventing, alleviating or treating oxidative stress in
a subject, at least about 200 mg Gotu kola powder is administered
to a subject daily using an herb-containing composition of the
invention.
[0087] Aloe vera
[0088] Aloe vera (common names: medicinal aloe, burn plant,
Barbados aloe, unguentine cactus) is a perennial plant; the strong,
fibrous root produces a rosette of fleshy basal leaves as in the
agave but considerably smaller that grows wild in East and South
Africa and also cultivated in the West Indies and other tropical
areas. Aloe contains anthraquinone glycosides, resins,
polysaccharides, sterols, gelonins, and chromones, which contribute
to the herbs medicinal properties. Aloe preparations, e.g., sap or
powder, are useful as emollients, purgatives, a vulnerary agent,
tonic, demulcent, vermifuge, antifungal, emmenagogue. Aloe
preparations are useful to treat burns, piles, sunburn, wrinkles,
headache, insect bites, skin irritations, cuts, ulcers, sores,
herpes, jaundice, bursitis, canker sores, sore gums, poison ivy,
inflammation, gastritis, and cancer. Aloe vera powder is
commercially available, e.g., Red Lion International Trading &
Brokerage Co. (Fullerton, Calif., USA).
[0089] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, from about 10 mg to about
4,000 mg Aloe vera powder is administered to a subject daily using
an herb-containing composition of the invention (See generally,
Table 2). In one embodiment of the method preventing, alleviating
or treating oxidative stress in a subject, from about 25 mg to
about 2,000 mg Aloe vera powder is administered to a subject daily
using an herb-containing composition of the invention. In one
embodiment of the method preventing, alleviating or treating
oxidative stress in a subject, from about 50 mg to about 1,000 mg
Aloe vera powder is administered to a subject daily using an
herb-containing composition of the invention. In one embodiment of
the method preventing, alleviating or treating oxidative stress in
a subject, at least about 200 mg Aloe vera powder is administered
to a subject daily using an herb-containing composition of the
invention.
[0090] Green Tea
[0091] Green tea extracts are useful in the compositions of the
present invention. In some embodiments of the compositions of the
invention, the Green tea extract is standardized for polyphenols.
For example, Green tea, 98% polyphenols containing 45% polyphenols
such as polyphenol (-)-epigallocatechin gallate (EGCG) is prepared
from the leaf of the tea herb Camellia sinensis. Polyphenols, e.g.,
EGCG, in green tea are useful to protective against certain
cancers, and they are also potent antioxidants. Green tea
preparations are useful to promote immune function and to prevent
and treat high cholesterol, heart disease, infection (e.g.,
Staphylococcus aureus infection, skin infection, bacterial
infection, viral infection), acne, aging, immune disorders, dental
caries, periodontitis, halitosis, dandruff, cancer, cardiovascular
disease (e.g., hypertension, thrombosis, arteriosclerosis),
diabetes, elevated blood glucose, diseases of the alimentary canal
and respiratory system, influenza hepatitis, liver disease. Green
tea extracts are commercially available, e.g., Hunan Kinglong
Bio-Resource Co., Ltd., (Xingsha, Changsha, Hunan, P. R.
China).
[0092] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, from about 5 mg to about
2,000 mg Green tea (98% polyphenols, 45% EGCG) is administered to a
subject daily using an herb-containing composition of the invention
(See generally, Table 2). In one embodiment of the method
preventing, alleviating or treating oxidative stress in a subject,
from about 10 mg to about 1,000 mg Green tea (98% polyphenols, 45%
EGCG) is administered to a subject daily using an herb-containing
composition of the invention. In one embodiment of the method
preventing, alleviating or treating oxidative stress in a subject,
from about 50 mg to about 500 mg Green tea (98% polyphenols, 45%
EGCG) is administered to a subject daily using an herb-containing
composition of the invention. In one embodiment of the method
preventing, alleviating or treating oxidative stress in a subject,
at least about 100 mg Green tea (98% polyphenols, 45% EGCG) is
administered to a subject daily using an herb-containing
composition of the invention.
[0093] Ginkgo biloba
[0094] Ginkgo biloba (common name: Maidenhair tree) is a dioecious
tree. Ginkgo leaf extracts have been shown to have a wide range of
biological activities. The leaf extract utilized in medicine is
standardized in a multi-step procedure designed to concentrate the
desired active principles from the plant. These extracts contain
approximately flavone glycosides (primarily composed of quercetin,
kaempferol, and isorhamnetin) and terpene lactones (ginkgolides A,
B, and C, and bilobalide). Other constituents typically include
proanthocyanadins, glucose, rhamnose, organic acids, D-glucaric
acid and ginkgolic acid (at most 5 ppm ginkgolic acids). The
complex extract itself, rather than a single isolated component, is
believed to be responsible for Ginkgo's biological activity. The
flavonoid complex can remove free radicals in the peripheral and/or
cerebral vascular systems, and inhibit lipid peroxidation. Ginko
biloba extract is commercially available, e.g., iHerb Inc.
(Monrovia, Calif., USA).
[0095] Some of the many valuable effects ginkgo has are stabilizing
cell membranes, reducing free radical damage, improving blood
circulation and enhancing oxygen and glucose use. Ginkgo is very
beneficial for the brain, nerves and blood vessels. It is useful to
improve short-term memory Ginkgo biloba has been used to improve
cerebral blood circulation and to protect the nerves against
damaging free radicals. Ginkgo leaf helps to maintain integrity and
permeability of cell walls by inhibiting lipid peroxidation of
membranes. Ginkgo extracts are useful to prevent or treat
age-related physical and mental deterioration (e.g., Alzheimer's
Disease and age-related dementia), cardiovascular disease, cerebral
vascular insufficiency and impaired cerebral performance,
congestive symptoms of premenstrual syndrome, allergies,
age-related vision loss, depression, Raynaud's disease, peripheral
vascular disease, intermittent claudication, vertigo, equilibrium
disorder, prevention of altitude sickness, tinnitus (ringing in the
ear), liver fibrosis, macular degeneration, asthma, graft
rejection, and immune disorders that induce toxic shock.
[0096] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, from about 5 mg to about
2,000 mg G. biloba leaf extract is administered to a subject daily
using an herb-containing composition of the invention (See
generally, Table 2). In one embodiment of the method preventing,
alleviating or treating oxidative stress in a subject, from about
10 mg to about 1,000 mg G. biloba leaf extract is administered to a
subject daily using an herb-containing composition of the
invention. In one embodiment of the method preventing, alleviating
or treating oxidative stress in a subject, from about 50 mg to
about 500 mg G. biloba leaf extract is administered to a subject
daily using an herb-containing composition of the invention. In one
embodiment of the method preventing, alleviating or treating
oxidative stress in a subject, at least about 200 mg G. biloba leaf
extract is administered to a subject daily using an herb-containing
composition of the invention.
[0097] N-Acetyl Cysteine
[0098] N-Acetyl Cysteine (NAC) is an acetylated form of the amino
acid cysteine. NAC is an antioxidant, an antitoxin and immune
support substance, and is found naturally in foods. There have been
several instances wherein NAC has been demonstrated to be an
antioxidant. NAC is commercially available, e.g., Doctor's Trust
Vitamins (Orlando, Fla., USA).
[0099] NAC reacts very slowly with superoxide or the hydrogen
peroxide free radicals. It can be seen from the rate constants that
GSH is a more effective antioxidant against the hydroxyl radical in
comparison to NAC (GSH K2=8.8.times.109, NAC K2=1.36.times.1010 at
a pH of 1.0). NAC will inhibit HOCl at physiological concentration
in a 3:1 ratio (respectively).
[0100] NAC is a precursor for glutathione, an important antioxidant
that protects cells against oxidative stress. In addition to
maintaining intracellular glutathione levels, NAC supplementation
has been shown to suppresses Human Immunodeficiency virus (HIV)
replication, to be protective against cell damage caused by
chemotherapy and radiation therapy, to be immune enhancing, to
protect against toxins as acetametaphen and other drugs, mercury,
lead, and others, and is mucolytic, that is, it breaks up mucus
seen in bronchoulmonary disease as cystic fibrosis, chronic
bronchitis, asthma, gastritis, heart attack, angina pectoris,
chronic obstructive pulmonary disease, prevention of kidney damage
during coronary angiography, Unverricht-Lundborg disease,
pseudoporphyria, and pneumonia. It has also been shown to offer
protection against the superoxide free radical in porcine aortic
endothelial cells and protects animals against paracetamol
hepatotoxicity.
[0101] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, from about 50 mg to about
5,000 mg N-acetyl cysteine is administered to a subject daily using
an herb-containing composition of the invention (See generally,
Table 2). In one embodiment of the method preventing, alleviating
or treating oxidative stress in a subject, from about 100 mg to
about 4,000 mg N-acetyl cysteine is administered to a subject daily
using an herb-containing composition of the invention. In one
embodiment of the method preventing, alleviating or treating
oxidative stress in a subject, from about 250 mg to about 2,000 mg
N-acetyl cysteine is administered to a subject daily using an
herb-containing composition of the invention. In one embodiment of
the method preventing, alleviating or treating oxidative stress in
a subject, at least about 500 mg N-acetyl cysteine is administered
to a subject daily using an herb-containing composition of the
invention.
[0102] Free radical damage on the clinical level will be determined
by the location of the preponderance of the oxidants generated, the
type of oxidants, and the generation of pathological
prostaglandins. The administration of compositions of the present
invention is dependent upon where pathological free radical
reactions are taking place. In one embodiment, the compositions of
the present invention are orally administered to a subject in need
thereof. Measuring the rate of the appearance of oxidation products
can assess the effectiveness of the compositions of the invention.
Effectiveness can also be monitored in patients by their clinical
progress.
[0103] In one aspect, the invention provides a method of increasing
the antioxidant activity level of a mammalian subject in need
thereof, by increasing the level of enzyme activity of at least one
enzyme, e.g., superoxide dismutase; catalase; and glutathione
peroxidase, by administering to the subject an effective amount of
an antioxidant-promoting composition of the invention, wherein the
increased enzyme activity decreases the tissue damage caused by
pathological free radicals. In one embodiment, the tissue damage
caused by pathological free radicals occurs in a mammalian subject
with a disease or condition selected from the group which includes,
e.g., inflammation; infection; atherosclerosis; hypertension;
cancer; radiation injury; neurological disease; neurodegenerative
disease; ischemia/reperfusion injury; aging; wound healing;
glutathione deficiency; acquired immunodeficiency syndrome; sickle
cell anemia; and diabetes mellitus. In one embodiment of the
method, the antioxidant-promoting composition is administered as an
oral dietary supplement.
[0104] In another embodiment the invention provides a method of
reducing the plasma level of c-reactive protein in a mammalian
subject, by administering to the subject an effective amount of an
antioxidant-promoting composition of the invention. In one
embodiment of the method, the antioxidant-promoting composition is
administered as an oral dietary supplement. In one embodiment of
the method, the plasma level of c-reactive protein decreases by at
least 10% or more in a 30 day period. In one embodiment of the
method, the plasma level of c-reactive protein decreases by at
least 20% or more in a 30 day period.
[0105] In another embodiment, the invention provides a method of
reducing the blood pressure in a mammalian subject, by
administering to the subject an effective amount of an
antioxidant-promoting composition of the invention. In one
embodiment of the method, the antioxidant-promoting composition is
administered as an oral dietary supplement.
[0106] ROS and Human Health
[0107] Our bodies are continuously exposed to free radicals and
other ROS, from both external sources (sunlight, other forms of
radiation, pollution) and generated endogenously; oxidative stress
and ROS-mediated tissue injury is a final common pathway for a
number of pathological processes. Oxidative stress results in
increased immune system activity, which leads to inflammation,
recruitment of more immune cells, and release of cytokines and
acute phase proteins that further exacerbate the stress on the
body.
[0108] Cytokines
[0109] It is postulated that in conditions where there is excessive
free radical production or infection (e.g. AIDS), there is a severe
alteration of interleukin-2 (IL-2) production, which secondarily
occurs due to glutathione (GSH) depletion. IL-2 is a glycoprotein,
which is produced in response to mitogens and antigenic stimuli; it
and other cytokines show a multiplicity of functions. Glutathione
levels regulate the alpha chain, the larger of the two IL-2
receptors. Decreasing GSH levels would decrease the affinity of
IL-2 to its corresponding receptors; consequently there would be a
compromise in the function of IL-2. It is postulated that
maintenance of GSH levels by the use of the compositions of the
present invention would allow IL-2 and its receptors to elicit the
normal immunological response for this particular interleukin.
[0110] Excessive oxidative stress results in amplified production
of TNF-alpha and IL-6. IL-6 initiates and encourages the production
of acute phase proteins such as c reactive protein, serum amyloid A
protein, fibrinogen, and mannan-binding lectin. IL-1, IL-6, and
TNF-alpha stimulate, for example, CRP synthesis by inducing hepatic
gene expression, which triggers a variety of inflammatory responses
and associated pathologies (see, Albert M A. The role of C-reactive
protein in cardiovascular disease risk. Curr Cardiol Rep 2000;
2(4):274-9). CRP is also a mediator of the complement system, part
of the innate immune response (see, Yuan G, Expression of C5aR
(CD88) of synoviocytes isolated from patients with rheumatoid
arthritis and osteoarthritis. Chin Med J (Engl). 2003 September;
116(9):1408-12). The complement system provides further stimulus of
TH1 and TH2 adaptive immune responses, which adds to the
inflammatory response.
[0111] Production of tumor necrosis factor-alpha (TNF-alpha) by
macrophages is stimulated by free radicals or oxidants (Chaudhri,
G. and Clark, I. A.: J Immunol, vol. 143, 1990-1294, No. 4, 1989).
TNF-alpha induces oxidant production by stimulating leukocytes,
releasing arachidonic acid from leukocytes and releasing lysosomes.
Therefore, enhancing plasma levels of antioxidants by administering
the compounds of the present invention would decrease the
production of TNF-alpha. It follows that, the maintenance/increase
in antioxidant potential by administering the compositions of the
present invention to a subject as described can prevent or treat
cytokine-mediated tissue injury.
[0112] Inflammation
[0113] Inflammation can arise from infective agents (e.g. virion),
trauma, chemical agents, immune reactions, metallic agents, and
ionizing or thermal agents. The sine qua non of inflammation is
heat, redness, edema, pain and loss of function (e.g., of the
surrounding tissue). In any type of inflammation, characteristic
inflammatory cells can be found, for example leukocytes,
eosinophils, and macrophages/macrocytes. Each of these cell types
produce radicals as part of a programmed response. Also as part of
that "programmed" response are the production of inflammatory
cytokines, such as TNF-alpha, CM-CSF and IL-2 and IL-6. These
particular cytokines promote the production of oxidants such as
nitric oxide and other reactive compounds. Oxidants are also
generated as a byproduct of prostaglandin (PG) production, which is
part of the propagation and amplification of the inflammatory
process. Platelets are also involved in the inflammatory process by
virtue of their ability to act as a plug (as in a clot); but also
due to their liberation of platelet activating factor (PAF). PAF
liberates arachidonic acid from leukocytes.
[0114] The production of prostaglandins is dependent upon the free
radical tone (or concentration) of the microenvironment and
metabolite synthesis. By decreasing the free radical tone and PG
free radical intermediate metabolites, it is postulated that the
pathological production of prostaglandins would be reduced, the
amplification effect that PGs have as a role in the inflammatory
process could be limited. Theoretically, either the lipooxygenase
limb or the cyclooxygenase limb of the prostaglandin pathway could
be affected by increased cellular antioxidants.
[0115] Free radicals or oxidants also have a plethora of different
effects on the tissue in which it occurs, e.g., membrane damage,
platelet adhesion, blood vessel intimal damage, etc. By increasing
the antioxidant levels in areas where inflammation is occurring, it
is postulated that the propagatory effect, tissue damage and
pathologic physiologic reactions would be curtailed as well. The
NF-KB transcription protein regulates the expression of a number of
genes for proteins and cytokines involved in the inflammatory
process (Baeuerle, P. A. and Baltimore, D.: Science, vol. 242,
October, 1988). The activity and affinity that the NF-KB protein
has for DNA is also regulated by GSH level (Staal, F. J. T, et al.:
Proc. Natl. Acad. Sci., USA, vol. 87, pp 9943-9947, December 1990;
Duh, E. J., et al.: Proc. Natl. Sci. Acad., USA, vol. 86, p
5974-5978, 1989). Enhancing levels of GSH decreases the activity
and binding of NF-KB to DNA. By enhancing GSH levels, those
cytokines and proteins involved in the inflammatory process would
be decreased. The maintenance/increase of antioxidant potential by
administering the compositions of the invention to a subject can,
therefore, prevent or treat inflammation-mediated injury.
[0116] One important marker for inflammation is c reactive protein
(CRP), a major acute phase response protein synthesized in the
liver in response to the elaboration of acute phase response
cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and
tumor necrosis factor alpha (TNF-alpha). Other associated acute
phase proteins include serum amyloid A protein, fibrinogen, and
mannan-binding lectin.
[0117] CRP concentrations are elevated in almost all inflammatory,
infectious, and neoplastic diseases. Specific conditions include
rheumatologic diseases (e.g., systemic lupus erythematosus,
Sjogren's syndrome, rheumatoid arthritis), vasculitides (e.g.,
Wegener's granulomatosis), and chronic infections (e.g.,
tuberculosis, endocarditis). Certain malignant neoplasms, such as
solid tumors, also may be associated with an elevated CRP level.
Because CRP has a long half-life, CRP levels correlate well to its
synthesis induced by persistent inflammation. Elevated levels of
CRP are also associated with cardiovascular diseases such as
endocarditis, angina pectoris, and myocardial infarction (see,
http://www.americanheart.org/presenterjhtml?identifier=4648 and
http://www.americanheart.org/presenterjhtml?identifier=3006541).
Certain pathogens have been linked to atherogenesis and the
development of clinically relevant coronary atherosclerosis. For
example, cytomegalovirus (CMV), Herpes simplex virus (HSV)
Chlamydia pneumoniae, and Helicobacter pylori have been associated
with coronary artery disease (CAD), through inducement of vascular
inflammation in addition to other mechanisms. This finding suggests
that CMV contributes to atherogenesis by provoking an inflammatory
response. CRP levels correlate with the clinical severity of CAD
and with coronary events in both the acute and subacute phases of
myocardial ischemia. Patients who are hospitalized for the
treatment of unstable angina and have CRP concentrations above 0.3
mg/dL have significantly more ischemic episodes in the hospital
than patients with lower CRP levels (see, Morrow D, Rifai N, Antman
E M, et al. C-reactive protein is a potent predictor of mortality
independently of and in combination with troponin T in acute
coronary syndromes: a TIMI 11A substudy. Thrombolysis in Myocardial
Infarction. J Am Coll Cardiol 1998; 31(7):1460-5). CRP
concentrations are significantly lower in patients with stable
angina pectoris than in those with unstable angina pectoris or an
acute coronary syndrome. Patients with chronic stable angina who
have stable, low CRP levels over time have fewer subsequent
cardiovascular events during follow-up (see, Bogaty P, Poirier P,
Simard S, et al. Biological profiles in subjects with recurrent
acute coronary events compared with subjects with long-standing
stable angina. Circulation 2001; 103(25):3062-8). On the other
hand, in patients with unstable angina pectoris, elevated CRP
levels are strong predictors of plaque instability.
[0118] Many trials have confirmed the association between high
levels of CRP and the risk of future coronary events such as MI and
sudden cardiac death. In the European Concerted Action on
Thrombosis and Disabilities study (Bolibar I, von Eckardstein A,
Assmann G, et al. Short-term prognostic value of lipid measurements
in patients with angina pectoris. The ECAT Angina Pectoris Study
Group: European Concerted Action on Thrombosis and Disabilities.
Thromb Haemost 2000; 84(6):955-60), elevation of mean CRP levels by
20% or more was found in patients after an MI. CRP levels are
higher in survivors of MI with or without a demonstrable coronary
lesion and increase further if other sites, such as peripheral
vasculature, also are involved. Hence, CRP levels may serve to
represent the inflammatory burden.
[0119] In the Monitoring Trends and Determinants in Cardiovascular
Disease trial (Koenig W, Sund M, Frohlich M, et al. C-reactive
protein, a sensitive marker of inflammation, predicts future risk
of coronary heart disease in initially healthy middle-aged men:
results from the MONICA (Monitoring Trends and Determinants in
Cardiovascular Disease) Augsburg Cohort Study, 1984 to 1992.
Circulation 1999; 99(2):237-42), a long-term prospective study of
cardiovascular risk, patients with the highest CRP levels had 2.6
times the risk of MI. In another study (Haverkate F, Thompson S G,
Duckert F. Haemostasis factors in angina pectoris; relation to
gender, age and acute-phase reaction. Results of the ECAT Angina
Pectoris Study Group. Thromb Haemost 1995; 73(4):561-7),
postinfarction angina occurred in only 14% of patients with a
normal CRP level. By comparison, 64% of patients admitted with high
CRP levels had evidence of postinfarction angina; nearly 42%
required revascularization, and 21% had recurrent MI.
[0120] In patients with MI, increased CRP concentration is
associated with the presence of complex angiographic lesions and
the need for revascularization (Moukarbel G V, Arnaout M S, Alam S
E. C-reactive protein is a marker for a complex culprit lesion
anatomy in unstable angina. Clin Cardiol 2001; 24(7):506-10).
Elevated CRP levels also may represent a biomarker for patients who
are most susceptible to reocclusion. In patients with stable CAD
who underwent stent implantation following angioplasty, CRP levels
increased over 96 hours in those with restenosis; in patients
without restenosis, CRP levels peaked at 48 hours and then
declined.
[0121] The normal serum concentration of CRP ranges from 3 mg/dL
(90th percentile of the general US population) to more than 200
mg/dL. Generally, the American Heart Association has suggested that
if hs-CRP level is lower than 1.0 mg/L, a person has a low risk of
developing cardiovascular disease; if hs-CRP is between 1.0 and 3.0
mg/L, a person has an average risk, and if hs-CRP is higher than
3.0 mg/L, a person is at high risk for cardiovascular disease.
[0122] Because these ranges are not sensitive for the values
required to determine cardiovascular risk in otherwise healthy
persons, investigators have developed new, modified techniques to
measure high-sensitivity CRP. The high-sensitivity CRP assay has
been shown to detect concentrations below 0.2 mg/mL and uses
labeled monoclonal or polyclonal anti-CRP antibodies in an
enzyme-linked immunosorbent assay (ELISA) or an immunofluorescent
assay (see, Rifai N, Ridker P M. High-sensitivity C-reactive
protein: a novel and promising marker of coronary heart disease.
Clin Chem 2001; 47(3):403-11).
[0123] Many conditions, activities, and medications affect levels
of C-reactive protein. Increased levels are seen following
allografts and graft occlusion, in connective tissue diseases
(e.g., lupus erythematosus, Wegener's granulomatosis), arthritis,
coronary artery disease, obesity, sepsis, in smokers, etc.
Decreased levels of CRP are seen in response to inhibitory
cytokines, exercise, and therapeutic doses of aspirin or
3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors
("statins"). Accordingly, the compositions of the present
invention, when administered to a subject having inflammation or
the related/resultant disorders listed, can reduce CRP levels
thereby reducing the inflammation and the severity of the
related/resultant disorders. Administration of these compositions
along with aspirin, naproxen, nambutome, other NSAID's or with
statins appears to have a synergistic or additive effect on
controlling inflammation and disease, depending on the particular
combination.
[0124] Atherosclerosis
[0125] Atherosclerosis remains the major cause of death and
premature disability in developed societies. Moreover, current
predictions estimate that by the year 2020 cardiovascular diseases,
notably atherosclerosis will become the leading global cause of
total disease burden, defined as the years subtracted from healthy
life by disability or premature death. Atherosclerosis is an
inflammatory vascular disease characterized by endothelial
activation, cellular influx, and production of mediators and
cytokines. This process leads to the formation of foamy macrophages
and atheromatous plaques and, finally, to atherothrombotic disease.
Atherosclerosis is associated with high morbidity and
mortality.
[0126] Atherosclerosis is a complex process that leads to heart
attack, stroke, and limb loss by the plugging of the arteries with
atherosclerotic plaque. This plaque is a form of oxidized fat. It
is now generally recognized that atherosclerosis is a chronic
inflammatory disease, characterized by overrecruitment of
leukocytes (monocytes and T-cells) to the site of inflammation.
Vascular injury in response to cardiovascular risk factors promotes
endothelial dysfunction, resulting in enhanced adhesion molecule
expression and secretion of pro-inflammatory cytokines and
chemokines. This, in turn, leads to adherence, migration and
accumulation of leukocytes within atherosclerotic lesions. The
recent findings on inflammatory processes involved in
atherosclerosis development provide important links between risk
factors and the mechanisms of atherogenesis. Thus, research
interest has increasingly focused on inflammatory biomarkers as
means of predicting the risk of future clinical events. Indeed,
elevated plasma levels of molecules such as soluble intercellular
adhesion molecule-1, interleukin-6 or C-reactive protein (CRP) have
been shown to represent inflammatory markers of future
cardiovascular risk. Among these, CRP has emerged as the most
powerful and accessible for clinical use (see, Steffens S.,
Inflammation and Atherosclerosis, Herz. 2004 December;
29(8):741-748).
[0127] CRP is a member of the pentraxin protein family, which is so
named because these proteins possess five identical subunits. CRP,
which is elaborated dramatically during acute inflammation,
augments the immune response to certain antigens, activates
complement, and increases the monocytic production of tissue
factors. CRP binds to phosphoryl choline on bacterial surfaces,
acting as an opsonin and playing a pivotal role in host defense.
Interestingly, CRP also appears to bind low-density lipoprotein
cholesterol (LDL-C) in vitro, which suggests a direct interaction
with the atherogenic lipids.
[0128] Inflammation in the vessels leads to the release of reactive
oxides and radicals from the immune cells. When radicals react with
lipids, the consequence is lipid peroxidation. While a number of
factors influence the development and severity of atherosclerosis,
a major factor is the ROS-mediated peroxidation of serum
low-density lipoproteins (LDLs). The dietary approach to the
prevention of heart disease and stroke is based partially on adding
dietary antioxidants to limit LDL oxidation, as well as decreasing
the intake of fat itself. These approaches already have made
significant inroads into the mortality from heart disease, but the
compositions of the present invention may offer a safe
pharmacological prevention in the future that is not as dependent
upon willpower as are diet and exercise. The maintenance/increase
of antioxidant potential by administering the compositions of the
present invention to a subject, therefore, can prevent or treat
cardiovascular disease, e.g., atherosclerosis.
[0129] Hypertension
[0130] The link between the elevation of the arterial blood
pressure and the production of vascular lesions remains an area of
inquiry. Recent evidence in the spontaneously hypertensive rat and
the salt dependent Dahl hypertensive strains indicate that there is
an excessive production of superoxide anion in microvascular
endothelium and in circulating leukocytes (Swei et al., Mechanisms
of Oxygen Free Radical Formation in Experimental Forms of
Hypertension, On-line Proceedings of the 5th Internet World
Congress on Biomedical Sciences '98 at McMaster University, Canada,
Presentation # SAswei0837, 1998). These studies examining the role
of the endothelial xanthine oxidase as a source for the oxidative
stress in the SHR and Dahl forms of hypertension and their
normotensive controls indicated that both models of hypertension
exhibit significantly elevated levels of XD and XO and enhanced
oxidative stress in both the arterial and venular segments of the
microcirculation compared with their respective normotensive
controls. The elevation of the oxidative stress and blood pressures
could be reduced significantly by blockade of XD and XO. Similarly,
the maintenance/increase of antioxidant potential by administering
the compositions of the present invention to a subject, therefore,
can prevent or treat cardiovascular disease, e.g.,
hypertension.
[0131] Membrane abnormalities in essential hypertensives (EH) are
known. The respiratory burst enzyme, NADPH oxidase is located in
the cell membrane of the neutrophil (PMNLs) and its activity is
important in generation of oxygen derived free radical (ROS). As
noted above, ROS have been implicated in vascular changes in
variety of conditions. Sagar and coworkers studied the status of
ROS and antioxidants in EH (Sagar et al., Mol Cell Biochem. 1992
April; 111(1-2):103-8).
[0132] Sagar and coworkers studied ten, age and sex-matched,
healthy controls (GpI) and 26 untreated EH (Gp IIA mild-8, Gp IIB
Moderate-8, Gp IIC Severe-10). After clinical examination and basic
laboratory evaluation of subjects, neutrophils isolated from their
blood were studied. Chemiluminescence (CL) emitted by PMNLs after
stimulation was measured (counts/min) in a luminometer and was
taken as measure of OFR production and thereby of NADPH oxidase
activity. The levels of antioxidants, superoxide dismutase (SOD)
and reduced glutathione (GSH), were also estimated.
Chemiluminescence was increased significantly (p less than 0.01) in
Gp IIC (243.04+/-24.9.times.10(3) counts per minute) as compared to
Gp IIA (2.80+/-1.87), Gp IIB (34.54+/-30.24) and Gp I (0.52+/-0.15)
and SOD was reduced significantly (p less than 0.05) in all EH (Gp
IIA 3.9+/-0.3 units per mg protein, Gp IEB 3.5+/-0.3 and Gp IIC
3.12+/-0.3) as compared to controls (4.1+/-0.2). Similarly GSH was
reduced (p less than 0.05) in EH (Gp IIA 11.2+/-1.7 mg per gm
protein, Gp IIB 8.5+/-1.1 and Gp IIC 6.6+/-0.3) as compared to Gp I
(13.5+/-2.5).
[0133] In addition, there is significant evidence that indicates
blood vessels thicken as a result of oxidative stress. Calcium
antagonists, especially the highly lipophilic amlodipine,
lacidipine and nisoldipine, are shown to possess antioxidant
properties. These drugs reduce the oxidation of LDL and its influx
into the arterial wall, and reduce atherosclerotic lesions in
animals. Platelet production of malondialdehyde, a marker of oxygen
free radical formation, is suppressed by amlodipine, lacidipine or
nifedipine in hypertensive patients. In the Regression Growth
Evaluation Statin Study (REGRESS), co-administration of calcium
antagonist, amlodipine or nifedipine with pravasatin caused a
significant reduction in the appearance of new angiographic
lesions. In the Verapamil in Hypertension and Atherosclerosis Study
(VHAS), verapamil was more effective than chlorthalidone in
promoting regression of thicker carotid lesions in parallel with a
reduction in the incidence of cardiovascular events. In the
Prospective Randomized Evaluation of the Vascular Effects of
Norvasc Trial (PREVENT), amlodipine slowed the progression of early
coronary atherosclerosis in patients with coronary artery disease.
In a subprotocol of the Intervention as a Goal in the Hypertension
Treatment (INSIGHT) study, nifedipine GITS significantly decreased
intima-media thickness as compared to co-amilozide
(hydrochlorothiazide+amiloride). Preliminary results of the
European Lacidipine Study on Atherosclerosis (ELSA) show that
lacidipine reduced the intima-media thickness progression rate as
compared to atenolol. Thus, selective calcium antagonists treat
hypertension and are potential antiatherosclerotic agents. (see,
Hernandez, R. H., Calcium antagonists and atherosclerosis
protection in hypertension, Am J Ther. 2003 November-December;
10(6):409-14). Likewise, the maintenance/increase of antioxidant
potential by administering the compositions of the present
invention to a subject, alone or in conjunction with calcium
channel antagonists therefore, can prevent or treat cardiovascular
disease, e.g., atherosclerosis, arterial lesions, intima-media
thickening, and hypertension. This is illustrated further in the
examples below.
[0134] Arthritis
[0135] Rheumatoid arthritis (RA) is a chronic multisystem disease
of unknown etiology. Although there are a variety of systemic
manifestations, the characteristic feature of RA is persistent
inflammatory synovitis, usually involving peripheral joints in a
symmetric distribution. The potential of the synovial inflammation
to cause cartilage destruction and bone erosions and subsequent
changes in joint integrity is the hallmark of the disease.
[0136] Direct correlation between the increases in TNF-alpha and
MMP-1 production and collagen degradation seen in the RA patient
suggests that collagenase cleavage of cartilage collagen is related
to the activities of TNF-alpha and MMP-1. The reduction in
cartilage type II collagen synthesis in early RA may contribute to
the developing pathology, since a lack of synthesis of this
molecule would inhibit maintenance of cartilage matrix. (see,
Fraser A., Turnover of type II collagen and aggrecan in cartilage
matrix at the onset of inflammatory arthritis in humans:
relationship to mediators of systemic and local inflammation.
Arthritis Rheum. 2003 November; 48(11):3085-95).
[0137] Deterioration of the joint is less likely to occur when the
patient CRP levels are consistently controlled (see, Dawes, P. T.,
Rheumatoid arthritis: treatment which controls the C-reactive
protein and erythrocyte sedimentation rate reduces radiological
progression. The British Journal of Rheumatology, Vol 25, 44-49).
CRP levels may also be helpful in following response to therapy in
rheumatic disorders, and may help to differentiate rheumatoid
arthritis (high levels of C-reactive protein) from uncomplicated
lupus (low levels of C-reactive protein). When used to evaluate
patients with arthritis, serum is the preferred specimen; there is
no reason to examine synovial fluid for C-reactive protein. CRP
levels, insulin sensitivity, HDL cholesterol, triglycerides and
hypertension are inter-related in RA patients, who typically
experience a markedly increased frequency of cardiovascular disease
(see, Dessein P H, Cardiovascular risk in rheumatoid arthritis
versus osteoarthritis: acute phase response related decreased
insulin sensitivity and high-density lipoprotein cholesterol as
well as clustering of metabolic syndrome features in rheumatoid
arthritis. Arthritis Res. 2002; 4(5):R5).
[0138] Generally, the first line of medical management of RA is the
use of nonsteroidal anti-inflammatory drugs (NSAIDs) and simple
analgesics to control the symptoms and signs of the local
inflammatory process. These agents are rapidly effective at
mitigating signs and symptoms, but they appear to exert minimal
effect on the progression of the disease. NSAIDs block the activity
of the Cox enzymes and therefore the production of prostaglandins,
prostacyclin, and thromboxanes. As a result, they have analgesic,
anti-inflammatory, and antipyretic properties. In addition, the
agents may exert other anti-inflammatory effects. Since these
agents are all associated with a wide spectrum of undesirable and
even toxic side-effects, the natural dietary supplement
compositions of the present invention provide a non-toxic
alternative to NSAIDs.
[0139] Although osteoarthritis (OA) is thought to derive from
defective chondrocyte metabolism and thus inherently lack the large
scale systemic response of rheumatoid arthritis, there is
increasing interest in the acute phase proteins in OA (see, Sowers
M., C-reactive protein as a biomarker of emergent osteoarthritis.
Osteoarthritis Cartilage. 2002 August; 10(8):595-601). Severity of
pain, is associated with hsCRP levels in patients with advanced OA
(see, Sturmer T., Severity and extent of osteoarthritis and low
grade systemic inflammation as assessed by high sensitivity C
reactive protein, Ann Rheum Dis. 2004 February; 63(2):200-5).
[0140] Accordingly, the maintenance/increase of antioxidant
potential in a mammal provides a method of ameliorating or
decreasing tissue degradation and inflammation seen in arthritis
(RA and OA). By administering the compositions of the present
invention to a subject as described above, the compositions can
therefore, prevent or treat OA and RA. The compositions may be
given with other pharmaceutical agents, e.g., Relafen and other
NSAID's or glucocorticoids (cortisone, dexamethasone, etc.) to
achieve a greater anti-inflammatory effect.
[0141] Cancer and Other Malignancies
[0142] Cancer and other malignancies all entail unconstrained cell
growth and proliferation based upon changes in the cell's genetic
information. In most cases, for example, one or more genes that
normally constrain cell growth and replication is/are mutated, or
otherwise inactivated. These genetic deficiencies correspond
directly with deletions and sequence changes in the genetic code,
resident in the cell's DNA. A frequently seen final common cause of
such DNA damage is free radical injury. Of the myriad injuries
sustained by our DNA on a daily basis, most are repaired by normal
DNA repair mechanisms within the cell, while some result in cell
death. Since such injuries are sporadic and distributed somewhat
randomly across the genome, most lethal DNA injuries are clinically
inconsequential, resulting in the loss of a few cells among
millions. However, when a single cell sustains an injury that
impairs growth regulation, it can proliferate disproportionately
and grow rapidly to dominate the cell population by positive
natural selection. The result is a tumor, frequently a malignant
one, where the constraint of growth and proliferation is
particularly deficient. Therefore, free radical injury to the
genetic material is a major final common pathway for
carcinogenesis.
[0143] An approach to creating anti-tumor therapeutics that appears
to have early success is aimed at preventing tumor induced
angiogenesis, thereby reducing blood supply to the tumor to prevent
growth and to kill the proliferating cells by starving them of
nutrients and oxygen. While most candidate therapeutics are direct
inhibitors of angiogenesis, other treatments are designed to
prevent initiation of the angiogenesis response. Tumor formation is
associated with localized inflammation, and increases of c-reactive
proteins (CRP), which are well known prognostic indicators of
patient survival (see, McKeown, D J., The relationship between
circulating concentrations of C-reactive protein, inflammatory
cytokines and cytokine receptors in patients with non-small-cell
lung cancer. Br J Cancer. 2004 Dec. 13; 91(12):1993-5). Elevated
CRP levels are seen in many types of cancer, see for example,
McArdle P A The relationship between interleukin-6 and C-reactive
protein in patients with benign and malignant prostate disease, Br
J Cancer. 2004 Nov. 15; 91(10):1755-7; Saddler, D., C-reactive
protein elevation and the risk of colorectal cancer, Gastroenterol
Nurs. 2004 September-October; 27(5):246-7; and Alexandrakis, MG The
relation between bone marrow angiogenesis and the proliferation
index Ki-67 in multiple myeloma, J Clin Pathol. 2004 August;
57(8):856-60.
[0144] CRP can significantly influence gene expression in the
vascular endothelium. CRP is upregulated by IL-6, and increases
expression of IL-8, ZF9, Activin A, MCP-1, EXT1, Cited2, PAI-1,
Fibronectin-1, Gravin, Connexin-43, and SORL-1, and decreases
expression of MAT2A, WRB, RCN1, TEB4, DNCL1 and Annexin A1 (see,
Wang, Q., Effect of C-Reactive Protein on Gene Expression in
Vascular Endothelial Cells, Am J Physiol Heart Circ Physiol. 2004
Dec. 9). Thus, CRP-responsive genes may have a broad functional
role in cell growth and differentiation, vascular remodeling and
solid tumor development. Inhibiting CRP would provide an additional
approach to current cancer therapies and may provide a prophylactic
anticancer effect by inhibiting inflammation and angiogenesis.
[0145] By administering the compositions of the present invention
to a subject as described above, the compositions can therefore,
prevent or treat cancers that respond to antioxidant treatments.
Such cancers include for example but not limited to, breast cancer
(see, Kline K, Vitamin E and breast cancer, J Nutr. 2004 December;
134(12 Suppl):3458S-3462S); lung cancer (see, Wright M E,
Development of a comprehensive dietary antioxidant index and
application to lung cancer risk in a cohort of male smokers. Am J
Epidemiol. 2004 Jul. 1; 160(1):68-76); ovarian cancer (see,
Anderson K, Differential response of human ovarian cancer cells to
induction of apoptosis by vitamin E Succinate and vitamin E
analogue, alpha-TEA. Cancer Res. 2004 Jun. 15; 64(12):4263-9); and
colon cancer (see, Al-Shaer MH., C-reactive protein and risk of
colon cancer. JAMA. 2004 Jun. 16; 291(23):2819). In fact,
colorectal carcinogenesis is associated with serious oxidative
stress gradual advancement of oxidative-antioxidative disorders is
followed by progression of colorectal cancer (see, Skrzydlewska E,
Lipid peroxidation and antioxidant status in colorectal cancer.
World J Gastroenterol. 2005 Jan. 21; 11(3):403-6).
[0146] The compositions may be given with other pharmaceutical
agents, e.g., glucocorticoids, camptothecins, mustard agents, and
other chemotherapy drugs, to achieve a greater anti-tumor response
and to control inflammation.
[0147] Radiation Injury
[0148] Radiation injury represents an important cause of
ROS-mediated disease. With respect to commonly encountered levels
of radiation, depending upon the situation, about two-thirds of the
sustained injury is mediated not by the radiation itself, but by
the ROS generated secondarily. This applies not only to the acutely
toxic forms of radiation injury, but the long-term, mutagenic (and
hence carcinogenic) effects as well.
[0149] An important clinical application of this principle is
encountered regularly in the treatment of cancer by radiation
therapy. Large tumors often outgrow their blood supplies and tumor
cells die within the center, despite being well oxygenated at the
periphery. Between these two regions is an area of tumor that is
poorly oxygenated, yet remains viable. Radiation therapy of such
tumors is particularly effective at the periphery, where an
abundant concentration of oxygen is available to form tumorcidal
ROS. The poorly oxygenated center is injured to a significantly
smaller degree. While the dead cells in the center don't survive
anyway, the poorly oxygenated, yet viable, cells between these two
areas can survive a safe dose of radiation therapy, and thereby
seed a later local recurrence of the tumor. This is a major reason
why many large tumors are treated by a combination of radiation
therapy (to kill the tumor at its advancing edges) and surgical
removal of the bulk of the tumor, including these particularly
dangerous remaining cells.
[0150] ROS can be generated within the cell not only by external
sources of radiation, but also within the body as a by-product of
normal metabolic processes. An important source of endogenous free
radicals is the metabolism of some drugs, pollutants, and other
chemicals and toxins, collectively termed xenobiotics. While some
of these are directly toxic, many others generate massive free
radical fluxes via the very metabolic processes that the body uses
to detoxify them. One example is the metabolism of the herbicide
paraquat. At one time, drug enforcement authorities used this
herbicide to kill marijuana plants. Growers realized they could
harvest the sprayed crop before it wilted, and still sell the
paraquat-laced product. Many who smoked this product subsequently
died of a fulminate lung injury. Fortunately, this approach has
been abandoned as a particularly inhumane way to solve the drug
problem.
[0151] While the paraquat story is a particularly striking example
of a metabolic mechanism of free radical toxicity, many commonly
encountered xenobiotics, including cigarette smoke, air pollutants,
and even alcohol are toxic, and often carcinogenic to a large
degree by virtue of the free radicals generated by their catabolism
within our bodies. Moreover, there is accumulating evidence that a
diet rich in fruits and vegetables, which are high in natural
antioxidants, and low in saturated fat (a particularly vulnerable
target for damage by ROS), reduces the risk of atherosclerosis and
cancer. The maintenance/increase of antioxidant potential by
administering the compositions of the present invention, therefore,
can prevent or treat atherosclerosis and cancer.
[0152] The maintenance/increase of antioxidant potential by
administering the compositions of the present invention to a
subject, therefore, can prevent or treat radiation-mediated
injury.
[0153] Neurological and Neurodegenerative Diseases
[0154] Neurological and neurodegenerative diseases affect millions
of Americans. These include depression, obsessive-compulsive
disorder, Alzheimer's, allergies, anorexia, schizophrenia, as well
as other neurological conditions resulting from improper modulation
of neurotransmitter levels or improper modulation of immune system
functions, as well as behavioral disorders such as ADD (Attention
Deficit Disorder) and ADHD (Attention Deficit Hyperactivity
Disorder). Oxidative stress links diverse neuropathological
conditions that include stroke, Parkinson's Disease, and
Alzheimer's Disease and has been modelled in vitro with various
paradigms that lead to neuronal cell death following the increased
accumulation of reactive oxygen species. For example, immortalized
neurons and immature primary cortical neurons undergo cell death in
response to depletion of the anti-oxidant glutathione, which can be
elicited by administration of glutamate at high concentrations.
[0155] A number of these diseases appear to have ROS toxicity as a
central component of their underlying mechanism of nerve cell
destruction, including, but not limited to, amyotrophic lateral
sclerosis (ALS, or Lou Gehrig's disease), Parkinson's disease, and
Alzheimer's disease. For example, Alzheimer's disease is a
neurodegenerative disorder associated with aging and cognitive
decline. Amyloid beta peptide (1-42) is a primary constituent of
senile plaques--a hallmark of Alzheimer's disease--and has been
implicated in the pathogenesis of the disease. Studies have shown
that methionine residue 35 of beta(1-42) may play a critical role
in Abeta(1-42)-mediated oxidative stress and neurotoxicity (see,
Boyd-Kimball D, Rodent Abeta(1-42) exhibits oxidative stress
properties similar to those of human Abeta(1-42): Implications for
proposed mechanisms of toxicity. J Alzheimer's Dis. 2004 October;
6(5):515-25).
[0156] Additionally, oxidative stress is associated with the
selective loss of dopaminergic neurons of the substantia nigra in
Parkinson's disease. The role of alpha synuclein as a potential
target of intracellular oxidants has been demonstrated by
identification of posttranslational modifications of synuclein
within intracellular aggregates that accumulate in PD brains, as
well as the ability of a number of oxidative insults to induce
synuclein oligomerization (see, Cole NB., Metal-catalyzed oxidation
of alpha synuclein: helping to define the relationship between
oligomers, protofilaments and filaments. J Biol Chem. 2004 Dec.
21).
[0157] Accordingly, the maintenance/increase of antioxidant
potential by administering the compositions of the present
invention to a subject, therefore, can prevent or treat
neurological and neurodegenerative diseases that have inflammation
and oxidative stress as causative or complicating factors.
[0158] Ischemia/Reperfusion Injury
[0159] When an organ is deprived of its blood supply (ischemia) it
is injured, not just by the temporary loss of oxygen, but also by
the ROS that are generated by reaction with the oxygen that is
reintroduced at reperfusion, when the blood supply is restored. In
some clinical situations, this injury can prevented by giving
antioxidants, sometimes even after the period of ischemia, but just
prior to reperfusion. For example, the preservation of kidneys,
livers, and other organs in solutions that contain antioxidants, as
well as other agents, is now routine prior to their
transplantation. Another example is the use of drugs that block the
function of free radical generating enzymes prior to stopping the
heart for cardiac surgery. These drugs help prevent reperfusion
injury when the heart is restarted and flow is restored. This
reperfusion injury mechanism also has been found to play an
important role in patients suffering from multiple organ failure
after trauma, massive surgery, or shock. Multiple organ failure is
now the leading cause of death in intensive care units, and
extensive efforts are under way to understand better how ROS
contribute to this syndrome.
[0160] Ischemia, which is low tissue oxygen saturation of a given
tissue, can occur in any organ system. All organs require a blood
supply in order to remain viable. The intact organ whose arterial
supply is compromised (either by partial or total occlusion) is
rendered ischemic (e.g., coronary artery occlusion, organs awaiting
transplantation, cerebral vascular accident, compartment syndrome,
etc.). There are reversible and irreversible histological,
physiological and biochemical changes which occur as a result of
ischemic injury to tissue. End stage ischemia is universal and
demonstrates necrosis. Demopoulos et al. (Fed. Proc. 32:1859-1861,
1973b) theorized that the necrosis observed in ischemic tissue was
due to oxidants generated by the uncoupling of the oxidative
phosphorylation chain in mitochondria. Zweir, et al., provided
direct evidence that free radical production resulted from ischemia
using electron spin resonance spectroscopy (Proc. Natl. Acad. Sci.
USA, vol. 84, pp: 1404-1407). In reperfusion studies Zweir showed
the alteration of one of the free radicals with the use of
superoxide dismutase (which eliminated superoxide). In ischemic
cardiac myocyte a depletion of ATP induces the release of
arachidonic acid and palmitic acid. Vitamin E (Massey, K. D. and
Burton, K. P.: Am. J. Physiol. 256 (Heart Circ. Physiol. 25):
H1192-H1199, 1989), vitamin E acetate and selenium selenite have
been used to protect tissue against free radicals, which have
occurred in ischemia. It is postulated that enhancement of tissue
antioxidants would eliminate the superoxide free radical, as well
as other oxidants that are not double produced as a result of
ischemia and prostaglandin metabolite production. The
maintenance/increase of antioxidant potential by administering the
compositions of the present invention to a subject, therefore, can
prevent or treat ischemia/reperfusion injury, e.g., brain ischemia
and heart ischemia (i.e., myocardial infarction).
[0161] Aging
[0162] Aging is a remarkably complex process that has managed to
remain relatively opaque to scientific understanding. There is now
evidence that aging is a series of processes, i.e., a series of
controlled mechanisms, and not just the passive accumulation of
wear and tear over the years. If aging is a series of processes,
some of these processes are potentially controllable, or at least
modifiable. One of the most important of these processes is
comprised of an accumulation of the molecular injuries that are
mediated by free radicals and other ROS. Recent studies indicate
that the therapeutic manipulation of ROS metabolism can actually
extend the total life span of mice to a significant degree. The
maintenance/increase of antioxidant potential by administering the
compositions of the present invention to a subject can, therefore,
prevent or treat age-mediate injury.
[0163] Burns/Wound Healing
[0164] Burn wounds to skin and other organs can occur by
ultraviolet radiation (UV), chemical agents, conductive or
convective heat, electrocution, etc. Burns can occur in lung
parenchyma by the inhalation of smoke or caustic gases (see section
on tissue injury). Burn wounds to the skin are graded as first,
second, and third degree burns (the most severe). It is postulated
that any burn wound produces tissue damage, largely by the
production of oxidants (Till, G. O.: Am J. Pathol. July; 1325(1):
195-202, 1989). Liposomes (artificial membranes) when exposed to UV
undergo peroxidation (Bose, B: Biotechnol Appl. Biochem, Oct., 12
(5): 557-61, 1990). It has been postulated that similar
peroxidation occurs in skin when it is exposed to UV radiation
(Somer, E.:Shape Magazine, p 33-35, November 1992; Hamanka, H.: J.
Dermatol, Oct. 17(10):595-8, 1990). Exposure of skin to UV varies
in intensity and length of exposure. Daily exposure to UV (e.g.,
sunlight) has been postulated to result in skin wrinkling.
Depending on the intensity and/or length of skin exposure to UV
light, first, second or third degree burns can result.
[0165] Hairless mice exposed to a single exposure of UV resulted in
a broad range decrease of antioxidants: glutathione, beta-carotene,
alpha-tocopherol. The enzyme activity of catalase and glutathione
reductase was also decrease (Fuchs, J.: J. Invest Dermatol, Dec.,
93(6): 769-73, 1989). These decreases in the concentration of
antioxidants and enzyme activity in skin due to UV exposure
supports the concept of the occurrence of free radicals in skin. It
is postulated that lipid peroxidation could be inhibited by an
enhancement of antioxidants in skin. Lipid peroxidation in
liposomes exposed to UV can be inhibited by placement of
beta-carotene or alpha-tocopherol in liposomes (Pelle, E.: Arch.
Biochem. Biophys. Dec. 283 (2): 234-40, 1990).
[0166] Excessive free radical production has been cited as a factor
in delayed wound healing (Yukie, N.: Dermatolgica, 179 (suppl 1):
101-106, 1989). The maintenance/increase of antioxidant potential
by administering the compositions of the present invention to a
subject of antioxidants would, therefore, ameliorate the effects of
pathologic oxidants and prostaglandin production as well as promote
wound healing in various skin injuries.
[0167] Tissue Injury and Degeneration
[0168] Tissue injury occurs as a result of an inflammatory focus
occurring in the area of a cell or an organ. For example plasma
oxidative stress occurs in patients with ulcerative colitis, and
omega-3 fatty acids are under study as free radical scavengers for
protecting the patients against the overall effect of oxidative
stress (see, Barbosa D S, Decreased oxidative stress in patients
with ulcerative colitis supplemented with fish oil omega-3 fatty
acids., Nutrition. 2003 October; 19(10):837-42). C-reactive protein
levels corresponded closely with clinical and pathological indices
of relapse, remission and response to therapy in patients with
Crohn's disease. Assay of serum C-reactive protein provides an
objective criterion of inflammatory activity, which may be useful
in the assessment, management and study of Crohn's disease (see,
Fagan, E. A., Serum levels of C-reactive protein in Crohn's disease
and ulcerative colitis. Eur J Clin Invest. 1982 August;
12(4):351-9). Accordingly, CRP levels may also be helpful in
following response to therapy for tissue injuries, and may help to
differentiate in Crohn's disease (high C-reactive protein) from
ulcerative colitis (low C-reactive protein).
[0169] Inflammation can occur due to a local inducement (e.g.
hepatitis) or due to an injury occurring to one organ in a remote
location and another discontiguous organ, which also sustains an
injury (e.g., severe burns occurring to skin (the first organ) with
subsequent injury to the lungs (the second organ)). In either case,
local or remote tissue injury is believed to be mediated by
activated leukocytes, which release oxidants. Oxidants released
from leukocytes react with cellular (organ) membranes (Fantone, J.
C. and Ward, P. A.: Am. J. of Path., vol. 107(3), P. 397-418,
1982). Repeated cellular membrane exposure to oxidants decreases
antioxidant levels, which increases their susceptibility to damage.
Increasing the levels of antioxidants in the extracellular and/or
intracellular, and/or the lipid-aqueous interface is postulated to
thwart oxidant damage to vital cellular structures. The
maintenance/increase of antioxidant potential by administering the
compositions of the invention to a subject can, therefore, prevent
or treat ROS-mediated tissue injury or other types of inflammatory
tissue degeneration.
[0170] Sepsis
[0171] Sepsis is characterized as a systemic infection by a
microorganism. Frequently it is fatal and if not fatal increases
the morbidity of the patient. In sepsis, red blood cells become
sticky and deformed (Baker, C. H., et al.: Circ. Shock 20:127-139,
1986; Powell, J., et al.: Critical Care Med., vol 19 (5), 1991),
which can lead to occlusion of the microvasculature. Cardiac output
is increased, but in the kidney, liver, and musculature blood flow
is decreased (Hurd, T. C., et al.: Archives of Surg., vol. 123,
1988). Evidence of free radical damage has been demonstrated in in
vitro and in vivo studies involving shock induced by endotoxins
(McKechnie, K., et al.: Circ. Shock 19: 429-439, 1986). Findings
include increased vascular permeability, damaged mitochondria,
disruption of calcium transport by the sarcoplasmic reticulum, and
the activation of the complement system (particularly C5a). In
septic infections, serum levels of acute phase proteins,
particularly CRP, are elevated, which increases activation of the
complement response as well as other cell mediated responses.
[0172] The maintenance/increase of antioxidant potential by
administration of the compositions of the present invention to a
subject can, therefore, prevent or treat sepsis-mediated
inflammatory cellular damage.
[0173] GSH Deficiency
[0174] Artificial depletion of glutathione interferes with normal T
cell function, particularly within the first 30-60 minutes of
activation (Fischman, C. M., et. al: The Journal of Immunology,
vol. 127(6), p 2257-2262, 1981; Hamilos, D. L. and Wedner, H. H.:
Journal of Immunology, vol. 135 (4), 1985). Glutathione deficient T
cells showed a decrease in thymidine incorporation and blast
transformation. The greater the depletion of glutathione the longer
it took cells to recover to normal levels. If cellular GSH
depletion was severe enough the cells never recovered to normal GSH
levels. Increased glutamate levels, which are found in AIDS
patients (Eck, H.-P. and Droge, W.: Bio. Chem. Hoppe-Seyler, vol.
370, pp 109-113), appear to inhibit the transport of cystine into
macrophages. Under normal circumstances cysteine is reduced to
cysteine by the macrophages. Cysteine is exported into the
microenvironment for the use of T cells for the ultimate conversion
to intracellular glutathione. T cells cannot utilize cystine. In
AIDS patients glutathione is depleted (Eck, H.-P, et al.: Biol.
Chem. Hoppe-Seyler, vol. 370, pp 101-108), which is postulated to
adversely effect T cell function. This scenario is believed to be
similar to the experimental studies, which demonstrated abnormal T
cell function as a result of artificial GSH depletion. The
maintenance/increase of antioxidant potential by administering the
compositions of the present invention to a subject can, therefore,
prevent or treat GSH deficiency.
[0175] AIDS
[0176] There is considerable evidence which indicates that HIV
infection and subsequently ARC/AIDS is by in large a free radically
mediated disease. This analysis can be made indirectly as judged by
the antioxidant levels in humans and their consequences on the
immune system. One of those antioxidants, glutathione (GSH), is
decreased as a result of HIV infecting the host. The GSH levels
continue to decrease as the disease progresses through ARC and
finally to AIDS. Micromolar changes in GSH levels have an untoward
effect on the function of T lymphocytes (which can be viewed as the
pivotal leader of the immune system). GSH shows a multiplicity of
uses in the immune system. Thiol concentrations (e.g. GSH) regulate
the replication of HIV genomic expression (Kalebic, T., et al.:
Proc. Natl. Acad. Sci., USA; 88: 986-90, 1991; Roeder, M., et al.,:
Porc. Natl. Acad. Sci., USA, vol. 87, p 4884-4888). Increasing the
concentrations of thiols (GSH, NAC, GSE (glutathione ester)) in
culture medium of U1 cell line (promonocytes) results in
suppression of viral assembly, HIV reverse transcriptase production
and viral replication. The maintenance of antioxidant potential by
administering the compositions of the invention to a subject can,
therefore, prevent or treat HIV-mediated injury.
[0177] Immunomodulation
[0178] There are numerous activators of leukocytes (e.g., exposure
to ingestible particles, certain soluble factors such as
complement, lectins, phorbol esters, etc.). A consequence of
leukocyte activation is the release of the MPO system
(H.sub.2O.sub.2+halide+myleoperoxidase) and other oxidants. The
more potent the stimulus of activation of leukocytes is, the
greater the release of oxidants and the greater the suppression of
T lymphocytic function. When activated leukocytes were combined
with T lymphocytes and catalase, there was no suppression of
lymphocytic function; monocytes (which contain enzymatic
antioxidants: glutathione peroxidase, catalase, myeloperoxidase)
were used in lieu of catalase, again there was no suppression
(Lipsky, P. E.: J. Clin, Invest. 73:53, 1984). Antibody production
by B lymphocytes showed a similar susceptibility to free radical
damage as did lymphocytes (El-Hag, A., et al.: J. of Immunol., vol.
136 (9), 1986). A following is a rank order for various lymphocytic
functions to free radical attack: immunoglobulin secreting cells
were the most sensitive (particularly to the MPO system); Natural
Killer cell activity, DNA synthetic responses to PHA and Con A were
intermediate; and the DNA response to PWM was the least
susceptible. Monocytes/macrophages have approximately 15-20 times
higher catalase content in comparison to lymphocytes (Meerhof, L.
J. and Roos, D.: J. Reticulendothel. Soc. 28: 419) and would
therefore be much less susceptible to oxidative damage. Lymphocytes
exposed to a free radical generating system demonstrate changes in
membrane characteristics: 63% decrease in E rosette formation, 44%
decrease in surface immunoglobulins and 90% decrease in cap
formation (Kraut, E. H. and Sagone, A. L.: J. of Lab. Clin. Med.,
November 1981, p 697-703). The maintenance/increase of antioxidant
potential by administering the compositions of the invention to a
subject can, therefore, prevent or treat immune damage by ROS.
[0179] Sickle Cell Anemia
[0180] Sickle cell anemia is a genetically determined disease.
Analysis of sickle cell patients RBC (HbS) demonstrates a number of
peculiarities of the membrane: frozen spectrin shell of
irreversibly sickled RBC, an abnormal orientation of the lipid
bilayer phospholipids, deficient calcium-ATPase, a propensity for
HbS RBCs to adhere to vascular endothelium, and oxidized thiol
groups on the HbS molecule. It is the characteristic of the
tendency of adherence to the vascular endothelium, which is the
likely primary pathogenesis of the disease, which is occlusion of
the microvasculature. Consequently, ischemic injury occurs to
organs (see section on ischemia). Additional evidence of free
radical damage to HbS is a deficiency of alpha-tocopherol,
increased amounts of malondialdehyde, and abnormal group cross
linking by malonadehyde. Superoxide anion can enter into
erthrocytes via anion channels, resulting in the formation of
methemoglobin and the ultimate lysis of erythrocytes (Weiss, S. J.:
J. Biol. Chem. 225: 9912-9917, 1980). Sickle RBCs spontaneously
generate sixty percent greater quantities of superoxide and
approximately 75% more hydrogen peroxide when compared with
controls (Hebbel, R. P., et al.: J. Clin. Invest., vol. 70, p.
1253-1259, 1982). Superoxide dismutase is increased by about 50%,
glutathione peroxidase and catalase were decreased by approximately
50% and 29% respectively. Glutathione and vitamin E levels were
significantly reduced. It is postulated that by increasing both
bone narrow and serum antioxidant levels that free radicals
produced by sickled RBCs would be markedly reduced. Accordingly,
maintenance/increase of antioxidant potential by administering the
compositions of the present invention is useful to prevent or treat
anemia-mediated injury.
[0181] Diabetes
[0182] Diabetes mellitus (DM) is a common disease affecting over
124 million individuals worldwide. DM is associated with high risk
of atherosclerosis and renal, neural, and ocular damage. Oxidative
stress results from a cell or tissue failing to detoxify the free
radicals that are produced during metabolic activity. Diabetes is
characterized by chronic hyperglycemia that produces dysregulation
of cellular metabolism. Vincent and coworkers have suggested that
diabetes overloads glucose metabolic pathways, resulting in excess
free radical production and oxidative stress (Vincent et al.,
Endocr Rev. August; 25(4):612-28 (2004). Vincent and coworkers have
presented evidence to support the idea that both chronic and acute
hyperglycemia cause oxidative stress in the peripheral nervous
system that can promote the development of diabetic neuropathy.
Proteins that are damaged by oxidative stress have decreased
biological activity leading to loss of energy metabolism, cell
signaling, transport, and, ultimately, to cell death. Examination
of the data from animal and cell culture models of diabetes, as
well as clinical trials of antioxidants, strongly implicates
hyperglycemia-induced oxidative stress in diabetic neuropathy.
Vincent et al., concluded that superior antioxidative therapies
remains essential for the prevention of neuropathy in diabetic
patients (Vincent et al., Endocr Rev., August; 25(4):612-28 (2004).
The maintenance/increase of antioxidant potential by administering
the compositions of the invention to a subject can, therefore,
prevent or treat diabetes-related, ROS-mediated tissue damage.
[0183] Pasaoglu and coworkers, investigated lipid peroxidation,
resistance of plasma and red blood cells to oxidation, and
antioxidant defense system in erythrocytes and sera in patients
with type 2 diabetes mellitus (Pasaoglu et al., Tohoku J Exp Med.,
July; 203(3):211-8 (2004). One group included newly diagnosed 20
patients and the other included 20 patients treated with oral
antidiabetic agents (OAD). Twenty healthy subjects served as
controls. Serum and red blood cell malondialdehyde (MDA),
glutathione (GSH), resistance to oxidation, and plasma thiol (total
--SH) levels were measured. In addition, glycated hemoglobin, serum
fructosamine, uric acid, total protein, total cholesterol,
triglyceride and glucose levels were determined. Although newly
diagnosed patients had higher serum and erythrocyte MDA levels than
those of controls, the highest levels of MDA were determined in
patients treated with OAD. MDA levels after exposing to oxidation
increased in OAD group more than in newly diagnosed patients. Total
--SH and erythrocyte GSH levels of the both diabetic groups were
lower than controls. These results showed that serum and
erythrocyte lipid peroxidation was increased in diabetic patients.
The sera of the patients showed a decreased resistance against
oxidation. Pasaoglu and coworkers proposed that the effect of
increased free radicals may be prevented by antioxidant systems in
early stages of type 2 diabetes but in advanced stages this
relationship is impaired owing to decreased antioxidant activity.
Decreased red blood cell GSH and serum total --SH levels may be due
to a compensation mechanism of the antioxidants. The
maintenance/increase of antioxidant potential by administering the
compositions of the invention to a subject can, therefore, prevent
or treat diabetes-related (e.g., diabetes type-2), ROS-mediated
tissue damage.
[0184] Administration of the antioxidant enzymes superoxide
dismutase (SOD) and catalase prevented destruction of islet
allograffs in NOD mice (Nomikos et al., Immunol Cell Biol 67:85-87
(1989)). Furthermore, the antioxidant probucol was shown to reduce
the diabetes incidence and to delay diabetes onset in the BB rats
(Drash A L et al., Am J Cardiol, 62:27 B-30B (1988)). Tabatabaie
and coworkers have demonstrated that chronic administration of the
free radical scavenger phenyl-N-tert-butyInitrone (PBN) inhibits
STZ-induced diabetes in mice (Tabatabaie et al., FEBS Lett,
407:148-152 (1997)). The low level of antioxidant enzymes such as
SOD, catalase, and glutathione peroxidase in the islets is another
indication that .beta.-cells are exceptionally vulnerable to
oxidative damage (Lenzen et al., Free Radical Biol. Med.,
20:463-466 (1996)).
[0185] ROS generation, evidenced by the formation of lipid
peroxidation products, is believed to be the ultimate cause of
cytokine-mediated death of .beta.-cells in isolated islets
(Rabinovitch et al., J. Clin. Endocrinol. Metab., 81:3197-3202
(1996)). Tabatabaie and coworkers recently demonstrate the
formation of free radicals in the pancreatic islets as a result of
cytokine treatment using EPR spectroscopy (Tabatabaie et al.,
Diabetes, August (2003)). Based on their studies, Tabatabaie and
coworkers concluded that free radicals have a role in the
pathogenesis of type 1 diabetes through .beta.-cell
cytokine-mediated free radical generation in the pancreatic Islets
(Tabatabaie et al., Diabetes, August (2003)). The
maintenance/increase of antioxidant potential by administering the
compositions of the invention to a subject can, therefore, prevent
or treat diabetes-related (e.g., diabetes type-1), ROS-mediated
tissue damage.
Pharmaceutical Compositions and Formulations
[0186] The compositions of the present invention can be used in
beverages, tonics, infusions, or foodstuffs alone, or in
combination with other dietary supplements or therapeutics. The
compositions of the invention can be used alone or further
formulated with pharmaceutically acceptable compounds, vehicles,
excipients or adjuvants with a favorable delivery profile, i.e.,
suitable for delivery to a subject. Such compositions typically
comprise the compositions of the invention and a pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal compounds, isotonic
and absorption delaying compounds, and the like, compatible with
pharmaceutical administration. Suitable carriers are described in
the most recent edition of Remington's Pharmaceutical Sciences, a
standard reference text in the field, which is incorporated herein
by reference. Preferred examples of such carriers or diluents
include, but are not limited to, water, saline, Ringer's solutions,
dextrose solution, and 5% human serum albumin. Liposomes and
non-aqueous vehicles such as fixed oils may also be used. The use
of such media and compounds for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or compound is incompatible with the active compound, use thereof
in the compositions is contemplated. Supplementary active compounds
can also be incorporated into the compositions.
[0187] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include oral, intravenous,
intraperitoneal, subcutaneous, intramuscular, intraarticular,
intraarterial, intracerebral, intracerebellar, intrabronchial,
intrathecal, topical, and aerosol route. The pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
Oral compositions generally include an inert diluent or an edible
carrier. They can be enclosed in gelatin capsules, caplets or
compressed into tablets. For the purpose of oral therapeutic
administration, the compositions of the invention can be
incorporated with one or more excipients and used in the form of
tablets, troches, or capsules. Oral compositions can also be
prepared using a fluid carrier for use as a mouthwash, wherein the
compound in the fluid carrier is applied orally and swished and
expectorated or swallowed. Pharmaceutically compatible binding
compounds, and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating compound such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening compound
such as sucrose or saccharin; or a flavoring compound such as
peppermint, methyl salicylate, or orange flavoring. Excipients can
also include, but are not limited to, e.g., calcium carbonate;
croscarmellose sodium; dicalcium phosphate; magnesium stearate;
microcrystalline cellulose; modified cellulose; silica; and stearic
acid.
[0188] The compositions of the invention can also be prepared as
pharmaceutical compositions in the form of suppositories (e.g.,
with conventional suppository bases such as cocoa butter and other
glycerides) or retention enemas for rectal delivery.
[0189] In one embodiment, the compositions of the invention are
prepared with carriers that will protect the compound against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions can also be used as pharmaceutically acceptable
carriers. These can be prepared according to methods known to those
skilled in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0190] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the composition of the invention and the
particular therapeutic effect to be achieved, and the limitations
inherent in the art of compounding such an active compound for the
treatment of individuals. The pharmaceutical compositions can be
included in a container, pack, or dispenser together with
instructions for administration.
[0191] The present compositions may be provided as a single dose,
however, it is preferred that the compounds be administered in
multiple doses. Particularly preferred are intermittent doses, such
as twice daily or once daily doses. However beneficial effects are
still seen with more sporadic intermittent doses, e.g., once every
36, 48, 60 or 72 hours, or once per week. Duration of treatment
will depend on the disorder being treated. For example, as
discussed herein, an average 24% decrease of serum CRP levels was
observed in human subjects treated intermittently for a 30 day
period with the present compositions. Further decrease in serum CRP
levels was observed to continue during the 90 day period of the
study. Extrapolating this information to a suggested dosage
regimen, one week may be sufficient to treat inflammation in post
operative surgical patients, whose serum CRP levels usually peak in
three days follow surgery, and naturally decline shortly
thereafter. Alternatively, chronic conditions such as arthritis or
atherosclerosis may require long term therapy over many years. A
medical professional will be able to determine dosage levels and
intervals in view of the teachings provided herein.
[0192] The invention is further defined by reference to the
following examples, which are not meant to limit the scope of the
present invention. It will be apparent to those skilled in the art
that many modifications, both to the materials and methods, may be
practiced without departing from the purpose and interest of the
invention.
EXAMPLES
Example 1
Herbal Compositions of the Invention Alter Antioxidant Enzymes and
Decrease Lipid Peroxidation in Wild Type C57BL/6J Mice
I. General Purpose and Study Design
[0193] The purpose of this study was to observe and effects of an
herbal composition of the present invention on normal 4 week old
wild type C57BL/6J mice. The exemplary herbal composition,
Protandim I, is a dietary supplement containing eight herbal
extracts, including, B. monniera extract, Milk Thistle extract
70-80%, Ashwagandha powder, Turmeric extract 95% curcumin, Gotu
kola powder, Aloe vera powder, Green tea (98% Polyphenols, 45%
EGCG), and Ginko biloba leaf extract and specifically detailed
below in Table 3.
TABLE-US-00003 TABLE 3 Composition of Protandim I. Percent Weight
Total Active Ingredient Amount Active Ingredients Bacopa monniera
extract, 45% 150 mg 12.77 bacosides Milk Thistle extract, 70-80%
275 mg 23.4 silymarin Ashwagandha powder 150 mg 12.77 Gotu kola
power 150 mg 12.77 Turmeric extract, 95% curcumin 75 mg 6.38 Green
tea 98% polyphenols, 45% 75 mg 6.38 EGCG Ginko biloba leaf extract
150 mg 12.77 Aloe vera powder 150 mg 12.77 Total 1175 mg 100
[0194] The composition was mixed with the powdered diet, then
pelleted. Mice were assigned to four groups as defined below in
Table 4, receiving dosages ranging from 0 to 1, 3, or 10 times the
anticipated human dosage. The estimated human dose for the present
studies was 1175 mg/day or 16 mg/kgbw/day. However, in other
embodiments of the invention the dosage to a subject is the amount
required to yield the desired antioxidant effect.
TABLE-US-00004 TABLE 4 Mice received the indicated amounts of
Protandim I Dose Protandim I No. of mice Diet .mu.g/gbw/day mg/kg
diet Group 1 7 Control 0 0 Group 2 8 1X 16 100 Group 3 7 3X 48 300
Group 4 8 10X 160 1000 TOTAL 30
[0195] The amount added was based on the assumption that the
average mouse consumes 5 g/d. The amount of Protandim I composition
received by each mouse in Group 2 was equivalent in mg/gbw to the
anticipated human dosage for a 70 kg person. Group 3 received a
3-fold higher dosage of Protandim I and Group 4 received a 10-fold
higher dosage of Protandim I. Mice were weighed at day 1 and day
23. After 23 days, the animals were sacrificed and tissues were
harvested for analysis. The end points measured included activities
of the major antioxidant enzymes SOD, CAT, and GPX in RBCs, liver,
and brain. In addition, the extent of lipid peroxidation was
assessed by measuring thiobarbituric acid reactive substances
(TBARS) in plasma, liver, and brain.
[0196] Thiobarbituric acid reactive substances, or TBARS, were
determined by the method of Ohkawa et al. (Ohkawa et al., Anal.
Biochem., 95: 351-358 (1979). The reaction mixture (total volume of
1 ml) contained 50 .mu.l of 8.1% sodium dodecyl sulfate, 0.375 ml
of 20% acetic acid and 0.375 ml 0.8% thiobarbituric acid, and 200
.mu.l of plasma or tissue homogenate supemate. The mixture was
heated in boiling water for 1 hr, cooled with tap water and
extracted with n-butanol/pyridine (15:1 v/v) by vortexing for 1-2
min. The mixture was then centrifuged at 500-1000 g for 10 min or
until a good aqueous-organic phase separation occurred. The organic
phase was removed and its absorbance at 532 nm was measured against
a reaction mixture blank. A standard curve was prepared with
1,1,3,3-tetramethoxypropane, and TBARS are reported as molar
equivalents.
[0197] Superoxide dismutase activity was determined by the method
of McCord and Fridovich. McCord et al., J. Biol. Chem., 244:
6049-6055 (1969).
[0198] Catalase was assayed by the method of Beers and Sizer. Beers
et al., J. Biol. Chem., 195, pp. 133-140 (1952). The disappearance
of peroxide is followed spectrophotometrically at 240 nm. The
incubation mixture (3 ml) contains 50 .mu.l of sample supernatant
in 0.05 M potassium phosphate (pH 7.0) and 0.02 M hydrogen
peroxide. The decrease in absorbance recorded at 240 nm for 2 min.
The rate of decrease in absorbance per min. is calculated from the
initial (45 sec.) linear portion of the curve.
[0199] The value of 0.0394 cm 2/.mu.mol is used as the extinction
coefficient of H.sub.2O.sub.2
[0200] One unit of catalase is defined as the amount of enzyme
which decomposes 1 .mu.mol of H.sub.2O.sub.2/min at 250.degree. C.
at pH 7.0 under the specified conditions.
[0201] Glutathione peroxidase was assayed as described by Carrillo
et al. (Carrillo et al., Life Sci., 48:517-521 (1991)).
II. Results
A. Assessment of Protandim I Toxicity
[0202] To assess any possible toxic effect of Protandim I
supplementation, animals were weighed at the beginning and at the
end of the study, and a percentage weight gain or loss was
calculated for each. The most general indication of toxicity is
"failure to thrive," which may manifest as a decreased rate of
growth, or as an actual weight loss. FIG. 1 shows that
supplementation actually caused modest but non-significant
increases in growth rate. The absence of toxicity was evidenced as
failure to thrive at any level of Protandim I supplementation, from
the anticipated human dosage (1.times.) to a level of ten times
that amount.
B. Protandim I Effect on SOD Activity
[0203] SOD activity was measured in the mice RBCs (FIG. 2), livers
(FIG. 3) and brains (FIG. 4) to assess the effect of Protandim I on
this enzyme. Animals supplemented with Protandim I showed a
dose-dependent increase in RBC SOD activity, as seen in FIG. 2. A
significant 25% increase was seen at the highest level of
supplementation. It should be noted that mature, circulating RBC do
not contain nuclei, and therefore are not capable of inducing new
synthesis of enzymes once they enter the circulation. RBC have a
circulating lifespan of 120 days. Thus, during the 23 day course of
the experiment about 20% of the RBC would have been replaced by
maturing reticulocytes from the bone marrow. As the older RBC are
diluted out by the newly produced cells, one may predict that the
increase in RBC SOD will proceed linearly with time until all cells
have been replaced at 120 days. Therefore, the predicted final RBC
SOD activities expected (after 120 days) would be about 16%, at
1.times., 44%, at 3.times., and 125% at 10.times. diet.
[0204] As detailed in FIG. 3, a dose-dependent increase in liver
SOD activity was observed. As shown in the figure, a significant
increase was seen at all levels of supplementation: 13% at
1.times., 23% at 3.times., and 45% at 10.times. diet. Also, a
significant increase in brain SOD activity was seen, but only at
the 3.times. level of Protandim I (FIG. 4). The percentage increase
was about 20%.
C. Protandim I Effect on CAT Activity
[0205] Animals supplemented with Protandim I showed little change
in CAT. In RBC, there appeared to be a small dose-dependent
increase, with an increase in the 10.times. group that approached
statistical significance (p=0.057) (FIG. 5). Based on the turnover
of RBC as discussed above, one can predict that the increase in RBC
CAT would proceed linearly with time until all cells have been
replaced at 120 days. Therefore, the predicted final RBC CAT
activities expected after 120 days might approach 10% at 1.times.,
20% at 3.times., and 40% at 10.times. diet.
[0206] Liver CAT was unchanged on 1.times. and 3.times. diets, but
showed a small non-significant decrease in activity on the
10.times. diet (FIG. 6). Brain normally contains very little
catalase, and the small amounts detected did not change at any diet
level.
D. Protandim I Effect on GPX Activity
[0207] Animals supplemented with Protandim I showed an unexpected
decrease in GPX activity. In RBC, there appeared to be a small
dose-dependent decrease, with a 13% decrease in the 10.times. group
that was not statistically significant (FIG. 7). Based on the
turnover of RBC as discussed above, one can predict that the
decrease in RBC GPX would proceed linearly with time until all
cells have been replaced at 120 days. Therefore, the predicted
decrease in RBC GPX activity expected after 120 days might approach
65% at 10.times. diet. There was a similar decrease in liver GPX
that appeared to be dose-dependent, with a 40% decrease in the
10.times. group that was statistically significant at p<0.004
(FIG. 8). Brain followed a similar pattern, with significant
decreases of 19% at 3.times. (p<0.03) and 23% at 10.times.
(p=0.01 (FIG. 9).
E. Protandim I Effect on Lipid Peroxidation (TBARS)
[0208] One objective of Protandim treatment is to decrease
oxidative stress. Our endpoint to assess oxidative stress in this
study was TBARS. Animals supplemented with Protandim I showed
dramatic and highly significant decreases in TBARS in all tissues
studied. In plasma, there was a dose-dependent decrease: 135%
decrease in animals on the 1.times. diet; 62# on the 3.times. diet;
and 75% on the 10.times. diet. The changes in the 3.times. and
10.times. groups had high statistical significance: p=0.004 and
p=0.0004, respectively (FIG. 10).
[0209] As shown in FIG. 11, in liver homogenates, there was a
similar dose-dependent decrease: a 34% decrease in animals on the
1.times. diet; 56% on the 3.times. diet; and 66% on the 10.times.
diet. The changes in this tissue had high statistical significance
at all there diet concentrations (FIG. 11). In brain homogenates,
the effect of Protandim I was even more striking: an 83% decrease
was seen on the 1.times. diet (p<0.004); 94% on the 3.times.
diet (p<0.0001; and 97% on the 10.times. diet (p<=0.0001)
(FIG. 12).
III. Summary of Protandim I's Effects
[0210] The present study established that herbal compositions of
the present invention, e.g., Protandim I (as defined in Table 3),
are a safe and effective way of decreasing oxidative stress. The
dosage defined as "1.times." (16 mg/kg body weight) seems perfectly
positioned for safety and efficacy. The major unexpected finding of
the study was that, of the three antioxidant enzymes measured, only
SOD was clearly induced. CAT was unaffected, and GPX levels
actually showed a significant decline. This study was based on
claims in the literature that seven of the eight herbal ingredients
upregulated SOD (Bhattacharya et al., Phytother, Res., 14:174-179
(2000); Soto et al., Comp Biochem. Physiol C. Toxicol. Pharmacol.,
136:205-212 (2003); Bhattacharya et al., Indian J. Exp. Biol.,
35:236-239 (1997); Bridi et al., Phytother. Res., 15:449-451
(2001); Luczaj et al., J. Toxicol. Environ. Health A, 67:595-606
(2004); Reddy et al., Food Chem., Toxicol., 32:279-283 (1994);
Singh et al., Phytomedicine, 7:209-219 (2000); Naidu et al., Indian
J. Exp. Biol., 40:894-900 (2002)), and that all eight upregulated
CAT, and GPX (Bhattacharya et al., Phytother, Res., 14:174-179
(2000); Soto et al., Comp Biochem. Physiol C. Toxicol. Pharmacol.,
136:205-212 (2003); Bhattacharya et al., Indian J. Exp. Biol.,
35:236-239 (1997); Bridi et al., Phytother. Res., 15:449-451
(2001); Luczaj et al., J. Toxicol. Environ. Health A, 67:595-606
(2004); Reddy et al., Food Chem., Toxicol., 32:279-283 (1994);
Singh et al., Phytomedicine, 7:209-219 (2000); Naidu et al., Indian
J. Exp. Biol., 40:894-900 (2002); Skukla et al., Phytother. Res.,
13:50-54 (1999)).
[0211] FIG. 13 summarizes the Protandim-induced changes in
antioxidant enzymes in blood, liver, and brain where clear and
distinctly different patterns emerge. SOD was induced in a
dose-dependent fashion, with the only departure from the pattern
being seen in brain with the 10.times. diet. As may be seen in FIG.
14, brain TBARS was, in fact, nearly totally eliminated on the
10.times. diet, suggesting that protection from oxidative stress
was maximal.
[0212] FIG. 14 summarizes the effect of each Protandim I diet level
on lipid peroxidation in each of the three tissues examined. There
was a strong dose-dependency apparent, with no suggestion of any
paradoxical increase in lipid peroxidation due to the
well-established bell-shaped curve seen with the effect of
increasing SOD concentration on rate of lipid peroxidation. This is
an important safety consideration. While it is clear that
increasing SOD will be beneficial to individuals experiencing high
oxidative stress, there was a possibility that increased SOD would
become problematic to healthy individuals with little oxidative
stress. This study, therefore, was designed to examine the ability
of Protandim I to decrease oxidative stress in young, normal mice,
without causing a paradoxical increase in oxidative stress at the
upper end of a ten-fold dosage range. Other compositions of the
invention likely share the same positive health benefits when
administered to a subject.
Example 2
Herbal Compositions of the Invention Alter Antioxidant Enzymes and
Decreased Lipid Peroxidation in Human Subjects
I. General Purpose and Design of Study
[0213] The purpose of this study was to observe the effects of
herbal compositions of the present invention on human subjects. An
exemplary dietary supplement containing five herbal extracts,
called Protandim II was administered to sixteen healthy human
subjects ranging in age from 20 to 78 years old. The composition of
Protandim II is shown below in Table 5.
TABLE-US-00005 TABLE 5 Composition of Protandim II. Percent Weight
Total Active Ingredient Amount Active Ingredients B. monniera
extract, 45% Bacosides 150 mg 22.2 Milk Thistle extract, 70-80% 225
mg 33.3 Silymarin Ashwagandha powder 150 mg 22.2 Turmeric extract,
95% Curcumin 75 mg 11.1 Green tea, 98% polyphenols, 45% 75 mg 11.1
EGCG Total 675 mg 99.9
[0214] Subjects were assigned to two groups as defined in Table
6.
TABLE-US-00006 TABLE 6 Protandim II Dosing of Human Subjects No. of
Age Daily supplement of subjects range Protandim II Group 1 12 20
to 78 675 mg Group 2 4 29 to 66 338 mg TOTAL 16
[0215] Group 1 had twelve subjects who received the full daily
Protandim II supplement of 675 mg in a single daily capsule for 120
days. At 0, 30, and 120 days, blood was taken by venipuncture for
analysis. Group 2 had four additional participants who received
one-half as much Protandim (i.e., half dose), or 338 mg in a single
daily capsule for 30 days.
II. Methods
[0216] The end points measured in tissue from human subjects
included activities of the major antioxidant enzymes SOD and CAT in
RBCs. The extent of lipid peroxidation was assessed by measuring
thiobarbituric acid reactive substances (TBARS) in plasma. In
addition, uric acid was measured in plasma because it is believed
to be an endogenous antioxidant of some importance, especially with
regard to scavenging and neutralizing the oxidant peroxynitrite. If
oxidative stress were decreased, one might expect a sparing effect
on plasma uric acid levels. High sensitivity CRP was monitored as
an indicator of inflammatory activity, and lipid profiles (total
cholesterol, LDL, HDL, and triglycerides) were assessed.
[0217] Thiobarbituric acid reactive substances, or TBARS, were
determined by the method of Ohkawa et al. (Ohkawa et al., Anal.
Biochem., 95: 351-358 (1979). The reaction mixture (total volume of
1 ml) contained 50 .mu.l of 8.1% sodium dodecyl sulfate, 0.375 ml
of 20% acetic acid and 0.375 ml 0.8% thiobarbituric acid, and 200
.mu.l of plasma or tissue homogenate supemate. The mixture was
heated in boiling water for 1 hr, cooled with tap water and
extracted with n-butanol/pyridine (15:1 v/v) by vortexing for 1-2
min. The mixture was then centrifuged at 500-1000 g for 10 min or
until a good aqueous-organic phase separation occurred. The organic
phase was removed and its absorbance at 532 nm was measured against
a reaction mixture blank. A standard curve was prepared with
1,1,3,3-tetramethoxypropane, and TBARS are reported as molar
equivalents.
[0218] Superoxide dismutase activity was determined by the method
of McCord and Fridovich. McCord et al., J. Biol. Chem., 244:
6049-6055 (1969).
[0219] Catalase was assayed by the method of Beers and Sizer. Beers
et al., J. Biol. Chem., 195, pp. 133-140 (1952). The disappearance
of peroxide is followed spectrophotometrically at 240 nm. The
incubation mixture (3 ml) contains 50 .mu.l of sample supernatant
in 0.05 M potassium phosphate (pH 7.0) and 0.02 M hydrogen
peroxide. The decrease in absorbance recorded at 240 nm for 2 min.
The rate of decrease in absorbance per min. is calculated from the
initial (45 sec.) linear portion of the curve.
[0220] The value of 0.0394 cm2/.mu.mol is used as the extinction
coefficient of H2O2
[0221] One unit of catalase is defined as the amount of enzyme
which decomposes 1 .mu.mol of H2O2/min at 250.degree. C. at pH 7.0
under the specified conditions.
[0222] High sensitivity C-reactive protein, uric acid, and lipid
profile analyses (total cholesterol, LDL, HDL, triglycerides) were
performed by the clinical chemistry laboratory of the University
Hospital/University of Colorado Health Sciences Center, Denver,
Colo.
[0223] Glutathione peroxidase was assayed as described by Carrillo
et al. (Carrillo et al., Life Sci., 48:517-521 (1991)).
III. Results
A. Assessment of Protandim II Toxicity or Side Effects
[0224] All subjects were instructed to report any suspected adverse
reaction or side effect (such as nausea, vomiting, headache,
gastrointestinal discomfort, diarrhea, constipation, itching, etc.)
to the investigators immediately, and to discontinue use of the
supplement. No such reactions or side effects were reported. No
toxicity or evidence of other unwanted pharmacological effects of
Protandim II were noted at either level of supplementation.
B. Effect of Protandim II on Lipid Peroxidation and TBARS
[0225] One objective of Protandim II treatment was to decrease
oxidative stress. Our endpoint to assess oxidative stress in this
study was TBARS, which measures a family of lipid peroxidation
products (mostly lipid hydroperoxides) which break down during the
analysis to yield malondialdehyde, which reacts with thiobarbituric
acid to yield the chromophore measured at 532 nm. The TBA assay has
been somewhat controversial, criticized by some for lack of
specificity because it yields higher values than gas
chromatographic methods specific for malondialdehyde. The ability
of the TBA test to collectively measure lipid peroxidation
products, including precursors that will continue to break down to
yield malondialdehyde, is a strength of the TBA assay. Gutteridge
et al., J. Appl. Biochem., 5:293-299 (1983); Liu et al., Anal.
Biochem. 245:161-166 (1997). It is clear that native fatty acids in
the absence of lipid peroxides do not undergo significant
peroxidation during the acid-heating stage of the TBA test.
Gutteridge et al., J. Appl. Biochem., 5:293-299 (1983).
Furthermore, the TBA test is the most widely used in the literature
to assess lipid peroxidation, enabling easy comparison with the
largest number of studies from other laboratories. In particular, a
recent study found plasma TBARS to be a predictor of cardiovascular
events in patients with established heart disease, independently of
traditional risk factors and inflammatory markers. Walter et al.,
J. Am. Coll. Cardiol., 44:1996-2002 (2004).
[0226] FIG. 15 (panel A and panel B) illustrates the age-related
increase in plasma TBARS in sixteen healthy human subjects ranging
in age from 20 to 78 years old, prior to supplementation with
Protandim II (solid circles). As shown in FIG. 15, panel A, while
there is substantial scatter around the linear regression line,
there is a strong correlation with age (R.sup.2=0.602) with the
oldest individuals showing values approximately three-fold higher
than the youngest individuals. After supplementation with Protandim
II for 30 days (675 mg/day, n=11), the values for plasma TBARS
declined as shown by the gray squares. The scatter is remarkably
less, and the correlation with age virtually disappears
(R.sup.2=0.082). After 30 days, the average TBARS concentration was
0.95.+-.0.04 .mu.M. Nine of these individuals were assayed after
120 days of supplementation (open circles), showing no further
change (0.99.+-.0.05 .mu.M; R.sup.2=0.012). All subjects showed
decreased TBARS after 30 days on Protandim II. The age-related
increase in lipid peroxidation products disappeared with Protandim
II supplementation. The changes were maintained at 120 days, with
results indistinguishable from those at 30 days.
C. Effect of Protandim II on SOD Activity
[0227] Subjects supplemented with Protandim II showed a
statistically significant increase of 22% (n=9, p=0.04) in
erythrocyte SOD activity after 120 days of supplementation, as seen
in FIG. 16. Erythrocyte SOD at day 0 was 378.+-.17 U/ml and
460.+-.18 U/ml by day 120. It should be noted that mature,
circulating erythrocytes do not contain nuclei, and therefore are
not capable of inducing new synthesis of enzymes once they enter
the circulation. Erythrocytes have a circulating lifespan of 120
days. Thus, during the 120 day course of the experiment 100% of the
red cells would have been replaced by maturing reticulocytes from
the bone marrow.
D. Effect of Protandim II on CAT Activity
[0228] Subjects supplemented with Protandim II showed a
statistically significant increase of 57% (n=9, p=0.001) in
erythrocyte CAT activity after 120 days of supplementation, as seen
in FIG. 17. Erythrocyte CAT at day 0 was 104,000.+-.8,000 U/ml and
163,000.+-.8,000 U/ml by day 120. The same considerations regarding
turnover and replacement of erythrocytes apply to catalase as
discussed above for SOD.
E. Effect of Protandim II on Select Blood Parameters
[0229] Subjects supplemented with Protandim II showed an increase
of 4.6% in plasma uric acid concentration after 30 days of
supplementation, but this increase did not achieve statistical
significance. Because uric acid serves as an endogenous
antioxidant, it was anticipated that uric acid levels might rise as
a result of increased SOD activity, which would lead to lower
levels of peroxynitrite production. Uric acid is thought to
scavenge the oxidant peroxynitrite.
[0230] Three subjects entered the study with elevated CRP levels,
there was a trend towards reduction of these levels with Protandim
II supplementation. As shown in FIG. 18, the CRP dropped an average
of 24% after 30 days of Protandim II supplementation. The CRP
levels continued to decline at 60-70 days, illustrating a decrease
of greater than average of 30%-33% decline in CRP levels over the
time tested. No significant changes were seen in total cholesterol,
LDL, HDL, or triglycerides.
F. Effect of Low-Dose Protandim II (338 mg/day)
[0231] To assess whether the suggested human supplement of 675
mg/day might be more than needed to achieve the desired reduction
in oxidative stress, four subjects were given a lower dose of 338
mg/day for 30 days. Blood was drawn from these individuals on day
0, 5, 12 and 30 to provide additional information regarding the
time required for the reduction in oxidative stress to manifest.
FIG. 19 shows that the response of plasma TBARS is fairly rapid,
with most of the change occurring by 5 to 12 days. FIG. 20 shows
that the lower dose of Protandim II was not quite as effective as
the full dose, lowering TBARS to an average value of 1.29.+-.0.14
.mu.M (n=4) versus 0.95.+-.0.04 .mu.M (n=11). Using a one-tailed
t-test, this difference was significant at p<0.03. This provides
reassurance of the appropriateness of the full recommended dose of
Protandim of 675 mg/day is not an excessive dose.
IV. Discussion
[0232] The present study established that herbal compositions of
the present invention, e.g., Protandim II (as defined in Table 5),
are a safe and effective way of decreasing oxidative stress in
healthy human subjects ranging in age from 20 to 78. The dosage
defined (675 mg/kg body weight) seems well-positioned for safety
and efficacy. The age-dependent increase in oxidative stress as
measured by lipid peroxidation was abolished. There was no evidence
that the subjects showing the lowest initial levels of oxidative
stress were in any way compromised by the modest elevations of SOD
and catalase that were achieved--an outcome considered remote but
theoretically possible due to our recognition that there is a
bell-shaped dose-response curve to SOD. That is, problems can
result from too much SOD as well as from too little. The results
from this study suggest that all subjects benefited from the
Protandim II-induced elevations of SOD and catalase activities.
[0233] The effects of Protandim II may go beyond direct induction
of the SOD and/or catalase genes. The antioxidant enzymes form a
system of mutual protection: superoxide inactivates both CAT and
GPX, while hydrogen peroxide inactivates the cytosolic SOD. McCord,
Free Radical Biol. Med., 4:9-14 (1988); Kono et al., J. Biol.
Chem., 257:5751-5754 (1982); Blum et al., Arch. Biochem. Biophys.,
240:500-508 (1985); Bray et al., Biochem. J., 139:43-48 (1974).
Thus, in a system experiencing substantial oxidative stress the
entire group of antioxidant enzymes may be subject to partial
inactivation by the unscavenged concentrations of superoxide and
hydrogen peroxide. If, under these conditions, SOD alone were
induced, the concentration of superoxide would decrease, allowing
partial recovery of the activities of CAT and GPX as they escape
from superoxide-mediated inactivation. Thus, it would appear that
all three enzymes were induced. This might be expected only when
starting under conditions of substantial oxidative stress. Under
normal conditions, there may be little inactivation of CAT and GPX
taking place, so the induction of SOD might have less effect on the
activities of the other two enzymes. Another factor to consider is
that 4-hydroxynonenal, a product of lipid peroxidation, serves to
induce the synthesis of GPX. Larini et al., Free Radic. Res.,
38:509-516 (2004). If the induction of SOD results in a lowering of
the rate of lipid peroxidation, then the concentration of
4-hydroxynonenal would fall and one might expect that less GPX
would be synthesized. This, in fact, is what we observed: SOD was
induced, lipid peroxidation was inhibited, and GPX activity fell.
Other compositions of the invention likely share the same positive
health benefits when administered to a subject.
Example 3
Herbal Compositions of the Invention Normalize Blood Pressure in
Human Subjects
[0234] Hypertension has been recognized as a multi-factorial trait
resulting from the effect of a combination of environmental and
genetic factors, including excess dietary salt or alcohol intake,
stress, age, genetics and family history, obesity, physical
inactivity, as well as high saturated fat diet. During the past few
years, however, a large amount of information has been collected on
the vascular inflammation, indicating that inflammation may involve
in the initiation as well as development of hypertension. Evidence
from animal models as well as patients, have indicated that
hypertension, an established major risk factor for coronary artery
disease, has been suggested to exert pro-inflammatory actions
through the increased expression of several mediators, including
leukocyte adhesion molecules, chemokines, specific growth factors,
heat shock proteins, endothelin-1, and angiotensin. Endothelial
dysfunction as well as increased serum levels of C-reactive protein
are observed in patients with hypertension (see, Li., J., Med
Hypotheses. Is hypertension an inflammatory disease? 2005;
64(2):236-240). Assessment of changes in gene expression associated
with increased arterial stiffness and gene polymorphisms that
increase the risk for vascular stiffening suggests that components
of the renin-angiotensin system, matrix metalloproteinases,
intracellular signaling, and extracellular matrix components may
all be involved in this process. Interventions aimed at these
targets may reduce vascular stiffness, lower systolic blood
pressure, decrease the prevalence of ISH, and improve outcomes for
patients (particularly older patients) with hypertension or other
CV conditions (see, Schiffrin E L., Vascular stiffening and
arterial compliance: implications for systolic blood pressure. Am J
Hypertens. 2004 December; 17(12 Pt 2):39S-48S). The compositions of
the present invention thus provide novel therapeutic strategies to
decrease the morbidity as well as mortality of hypertension, and
alleviated hypertensive target organ damage.
[0235] Subject #1 was a 50 year old man whose blood pressure
readings prior to administration of a composition of the invention
ranged from 140 to 150 systolic over 90 to 100 diastolic. Following
daily ingestion of 1500 mg of a composition containing B. monniera
extract, Milk Thistle extract 70-80%, Ashwagandha powder, Turmeric
extract 95%, Gotu kola powder, Aloe vera powder, Green tea (98%
Polyphenols 45% EGCG), and Ginko biloba leaf extract for 14 days,
the subjects blood pressure was measured as 126 systolic over 74
diastolic. The approximate daily dosage of each of the components
of the herb-containing composition was as follows: B. monniera
extract (200 mg), Milk Thistle extract 70-80% (300 mg), Ashwagandha
powder (200 mg), Turmeric extract (95%) (100 mg), Gotu kola powder
(200 mg), Aloe vera powder (200 mg), Green tea (98% Polyphenols 45%
EGCG) (100 mg), and Ginko biloba leaf extract (200 mg).
[0236] Subject # 2 was a 44 year old woman whose blood pressure
readings prior to any treatment were approximately 165 systolic
over 113 diastolic to 160 systolic over 103 diastolic. The subject
began taking ATENOLOL 25 mg/day (9 months). The ATENOLOL medication
reduced the subject's blood pressure to 142 systolic over 98
diastolic to 135 systolic over 96 diastolic, however, the subject's
diastolic measurement never got down below 91 (the lowest on
ATENOLOL). Following daily ingestion of 1,500 mg of an
herb-containing composition containing B. monniera extract, Milk
Thistle extract 70-80%, Ashwagandha powder, Turmeric extract 95%,
Gotu kola powder, Aloe vera powder, Green tea (98% Polyphenols 45%
EGCG), and Ginko biloba leaf extract for 21 days (while ATENOLOL
treatment was continued), the subjects blood pressure was measured
as 136 systolic over 70 diastolic. Thus, a drop in diastolic
pressure of more than 20 mm was observed during this period (see
FIG. 21, panel A). Thereafter, blood pressure remained constant as
both treatments continued. The approximate daily dosage of each of
the components of the herb-containing composition was as follows:
B. monniera extract (200 mg), Milk Thistle extract 70-80% (300 mg),
Ashwagandha powder (200 mg), Turmeric extract (95%) (100 mg), Gotu
kola powder (200 mg), Aloe vera powder (200 mg), Green tea (98%
Polyphenols 45% EGCG) (100 mg), and Ginko biloba leaf extract (200
mg).
[0237] Subject #3 was a 49 year old man whose blood pressure
readings prior to administration of a composition of the invention
was 135 systolic over 78 diastolic. Following daily ingestion of
1500 mg of a composition containing B. monniera extract, Milk
Thistle extract 70-80%, Ashwagandha powder, Turmeric extract 95%,
Gotu kola powder, Aloe vera powder, Green tea (98% Polyphenols 45%
EGCG), and Ginko biloba leaf extract for 7 days, the subjects blood
pressure was measured as 117 systolic over 75 diastolic. The
approximate daily dosage of each of the components of the
herb-containing composition was as follows: B. monniera extract
(200 mg), Milk Thistle extract 70-80% (300 mg), Ashwagandha powder
(200 mg), Turmeric extract (95%) (100 mg), Gotu kola powder (200
mg), Aloe vera powder (200 mg), Green tea (98% Polyphenols 45%
EGCG) (100 mg), and Ginko biloba leaf extract (200 mg).
[0238] Subject # 4 was a 59 year old man whose blood pressure
readings prior to any treatment were approximately 128 systolic
over 75 diastolic. Following daily ingestion of 1000 mg of a
composition containing B. monniera extract, Milk Thistle extract
70-80%, Ashwagandha powder, Turmeric extract 95%, Gotu kola powder,
Aloe vera powder, Green tea (98% Polyphenols 45% EGCG), and Ginko
biloba leaf extract for 21 days, the subject's blood pressure was
measured as 125 systolic over 61 diastolic. Thus, a drop in
diastolic pressure of about 14 mm was observed during this period
(see FIG. 21, panel B). After 21 days, blood pressure remained
constant as treatment continued. The approximate daily dosage of
each of the components of the herb-containing composition was as
follows: B. monniera extract (134 mg), Milk Thistle extract 70-80%
(200 mg), Ashwagandha powder (134 mg), Turmeric extract (95%) (67
mg), Gotu kola powder (134 mg), Aloe vera powder (134 mg), Green
tea (98% Polyphenols, 45% EGCG) (67 mg), and Ginko biloba leaf
extract (134 mg).
Example 4
Compositions of the Invention Prevents or Alleviates Migraine
Headaches in Human Subjects
[0239] Migraines afflict about 24 million people in the United
States. They may occur at any age, but usually begin between the
ages of 10 and 40 and diminish after age 50. Some people experience
several migraines a month, while others have only a few migraines
throughout their lifetime. Approximately 75% of migraine sufferers
are women.
[0240] A migraine is a throbbing or pulsating headache that is
often one sided (unilateral) and associated with nausea; vomiting;
sensitivity to light, sound, and smells; sleep disruption; and
depression. Attacks are often recurrent and tend to become less
severe as the migraine sufferer ages. Migraines are classified
according to the symptoms they produce. The two most common types
are migraine with aura and migraine without aura. Less common types
include the following: Basilar artery migraine; Carotidynia;
Headache-free migraine; Opthalmoplegic migraine; Status
migraine
[0241] The cause of migraine is unknown. The condition may result
from a series of reactions in the central nervous system caused by
changes in the body or in the environment. There is often a family
history of the disorder, suggesting that migraine sufferers may
inherit sensitivity to triggers that produce inflammation in the
blood vessels and nerves around the brain, causing pain.
[0242] A trigger is any stimulus that initiates a process or
reaction. Commonly identified migraine triggers include the
following: Alcohol (e.g., red wine); environmental factors (e.g.,
weather, altitude, time zone changes); foods that contain caffeine
(e.g., coffee, chocolate); monosodium glutamate (MSG; found in
Chinese food); and nitrates (e.g., processed foods, hot dogs);
glare; hormonal changes in women; hunger; lack of sleep,
medications (over-the-counter and prescription); perfume;
stress.
[0243] In the present study, a female subject (woman) who routinely
suffers migraine headaches was administered a composition of the
invention to assess the affect of the composition on the incidence
of her migraine headaches. The subject ingested a composition
containing B. monniera extract, Milk Thistle extract 70-80%,
Ashwagandha powder, Turmeric extract 95%, Gotu kola powder, Aloe
vera powder, Green tea (98% Polyphenols 45% EGCG), and Ginko biloba
leaf extract for 30 days. The approximate daily dosage of each of
the components of the herb-containing composition was as follows:
B. monniera extract (134 mg-200 mg), Milk Thistle extract 70-80%
(200 mg-300 mg), Ashwagandha powder (134 mg-200 mg), Turmeric
extract (95%) (67 mg-100 mg), Gotu kola powder (134 mg-200 mg),
Aloe vera powder (134 mg-200 mg), Green tea (98% Polyphenols 45%
EGCG) (67 mg-100 mg), and Ginko biloba leaf extract (134 mg-200
mg). Following the initiation of administration of the composition
of the invention, the subject has not had a migraine headache. The
composition of the invention is therefore useful to prevent or
alleviate migraine headaches in a subject.
[0244] Similarly, the compositions of the present invention are
useful to prevent or treat headaches in a subject associated with
high altitude (e.g., acute mountain sickness (AMS)). In the context
of a recent ascent, a headache, with any one or more of the
following symptoms above 2500 meters (8000 feet) qualifies a
subject for the diagnosis of AMS: loss of appetite, nausea, or
vomiting; fatigue or weakness; dizziness or light-headedness;
difficulty sleeping; confusion; staggering gait. The compositions
of the present invention are also useful to prevent or treat
headaches, both acute and chronic.
Example 5
Embodiments of the Herb-Containing Composition of the Invention
[0245] In one embodiment of the method preventing, alleviating or
treating oxidative stress in a subject, an herb-containing
composition containing B. monniera extract, Milk Thistle extract,
70-80% Silymarin, Ashwagandha powder, Green tea, 98% polyphenols,
45% EGCG, and Turmeric extract, and 95% Curcumin is administered at
least daily to a subject. In one embodiment, the herb-containing
composition of the invention contains B. monniera extract, 45%
bacosides, Milk Thistle extract, 70-80% Silymarin, Ashwagandha
powder, Green tea, 98% polyphenols, 45% EGCG, and turmeric extract,
95% curcumin in concentrations as detailed below in Table 7.
TABLE-US-00007 TABLE 7 Composition of Protandim II Weight Percent
(wt %) Total Active Ingredient Quantity Active Ingredients Bacopa
monniera, extract 150 mg 22.2 45% bacosides Milk Thistle extract,
225 mg 33.3 70-80% silymarin Ashwagandha powder 150 mg 22.2 Green
tea, 98% 75 mg 11.1 polyphenols, 45% EGCG Turmeric extract, 95% 75
mg 11.1 curcumin Total 675 mg 99.9
[0246] In one embodiment, the herb-containing composition of the
invention contains from about 5 wt % to about 50 wt % B. monniera
extract (45% bacosides) of the total weight of active ingredients.
In one embodiment, the herb-containing composition of the invention
contains from about 10 wt % to about 30 wt % B. monniera extract
(45% bacosides) of the total weight of active ingredients. In one
embodiment, the herb-containing composition of the invention
contains at least about 22 wt % B. monniera extrac (45% bacosides)
of the total weight of active ingredients.
[0247] In one embodiment, the herb-containing composition of the
invention contains from about 5 wt % to about 60 wt % milk thistle
extract (70%-80% silymarin) of the total weight of active
ingredients. In one embodiment, the herb-containing composition of
the invention contains from about 10 wt % to about 50 wt % milk
thistle extract (70%-80% silymarin) of the total weight of active
ingredients. In one embodiment, the herb-containing composition of
the invention contains at least about 33 wt % milk thistle extract
(70%-80% silymarin) of the total weight of active ingredients.
[0248] In one embodiment, the herb-containing composition of the
invention contains from about 5 wt % to about 50 wt % Ashwagandha
powder of the total weight of active ingredients. In one
embodiment, the herb-containing composition of the invention
contains from about 10 wt % to about 30 wt % Ashwagandha extract of
the total weight of active ingredients. In one embodiment, the
herb-containing composition of the invention contains at least
about 22 wt % Ashwagandha extract of the total weight of active
ingredients.
[0249] In one embodiment, the herb-containing composition of the
invention contains from about 2.5 wt % to about 25 wt % turmeric
extract (95% curcumin) of the total weight of active ingredients.
In one embodiment, the herb-containing composition of the invention
contains from about 5 wt % to about 15 wt % turmeric extract (95%
curcumin) of the total weight of active ingredients. In one
embodiment, the herb-containing composition of the invention
contains at least about 11 wt % turmeric extract (95% curcumin) of
the total weight of active ingredients.
[0250] In one embodiment, the herb-containing composition of the
invention contains from about 2.5 wt % to about 25 wt % green tea
(98% polyphenols, 45% EGCG) of the total weight of active
ingredients. In one embodiment, the herb-containing composition of
the invention contains from about 5 wt % to about 15 wt % green tea
(98% polyphenols, 45% EGCG) of the total weight of active
ingredients. In one embodiment, the herb-containing composition of
the invention contains at least about 11 wt % green tea (45%
polyphenols) of the total weight of active ingredients.
[0251] In some embodiments of the herb-containing composition of
the present invention, the composition comprises the active
ingredients as summarized above in Table 7 as well as Gotu kola
powder, Ginko biloba leaf extract and Aloe vera powder as detailed
below in Table 8.
TABLE-US-00008 TABLE 8 Composition of Protandim I. Percent Weight
Total Active Ingredient Amount Active Ingredients Bacopa monniera
extract, 45% 150 mg 12.77 bacosides Milk Thistle extract, 70-80%
275 mg 23.4 silymarin Ashwagandha powder 150 mg 12.77 Gotu kola
power 150 mg 12.77 Turmeric extract, 95% curcumin 75 mg 6.38 Green
tea 98% polyphenols, 45% 75 mg 6.38 EGCG Ginko biloba leaf extract
150 mg 12.77 Aloe vera powder 150 mg 12.77 Total 1175 mg 100
[0252] In one embodiment, the herb-containing composition of the
invention contains from about 5 wt % to about 50 wt % B. monniera
extract (45% bacosides) of the total weight of active ingredients.
In one embodiment, the herb-containing composition of the invention
contains from about 10 wt % to about 30 wt % B. monniera extract
(45% bacosides) of the total weight of active ingredients. In one
embodiment, the herb-containing composition of the invention
contains at least about 22 wt % B. monniera extract (45% bacosides)
of the total weight of active ingredients. In one embodiment, the
herb-containing composition of the invention contains at least
about 12 wt % B. monniera extract (45% bacosides) of the total
weight of active ingredients.
[0253] In one embodiment, the herb-containing composition of the
invention contains from about 5 wt % to about 60 wt % milk thistle
extract (70%-80% silymarin) of the total weight of active
ingredients. In one embodiment, the herb-containing composition of
the invention contains from about 10 wt % to about 50 wt % milk
thistle extract (70%-80% silymarin) of the total weight of active
ingredients. In one embodiment, the herb-containing composition of
the invention contains at least about 33 wt % milk thistle extract
(70%-80% silymarin) of the total weight of active ingredients.
[0254] In one embodiment, the herb-containing composition of the
invention contains at least about 23 wt % milk thistle extract
(70%-80% silymarin) of the total weight of active ingredients.
[0255] In one embodiment, the herb-containing composition of the
invention contains from about 5 wt % to about 50 wt % Ashwagandha
powder of the total weight of active ingredients. In one
embodiment, the herb-containing composition of the invention
contains from about 10 wt % to about 30 wt % Ashwagandha extract of
the total weight of active ingredients. In one embodiment, the
herb-containing composition of the invention contains at least
about 22 wt % Ashwagandha extract of the total weight of active
ingredients. In one embodiment, the herb-containing composition of
the invention contains at least about 12 wt % Ashwagandha extract
of the total weight of active ingredients.
[0256] In one embodiment, the herb-containing composition of the
invention contains from about 2.5 wt % to about 25 wt % turmeric
extract (95% curcumin) of the total weight of active ingredients.
In one embodiment, the herb-containing composition of the invention
contains from about 5 wt % to about 15 wt % turmeric extract (95%
curcumin) of the total weight of active ingredients. In one
embodiment, the herb-containing composition of the invention
contains at least about 11 wt % turmeric extract (95% curcumin) of
the total weight of active ingredients. In one embodiment, the
herb-containing composition of the invention contains at least
about 6 wt % turmeric extract (95% curcumin) of the total weight of
active ingredients.
[0257] In one embodiment, the herb-containing composition of the
invention contains from about 2.5 wt % to about 25 wt % green tea
(98% polyphenols, 45% EGCG) of the total weight of active
ingredients. In one embodiment, the herb-containing composition of
the invention contains from about 5 wt % to about 15 wt % green tea
(98% polyphenols, 45% EGCG) of the total weight of active
ingredients. In one embodiment, the herb-containing composition of
the invention contains at least about 11 wt % green tea (45%
polyphenols) of the total weight of active ingredients. In one
embodiment, the herb-containing composition of the invention
contains at least about 6 wt % green tea (45% polyphenols) of the
total weight of active ingredients.
[0258] In one embodiment the composition, the Gotu kola powder is
present at a concentration from at least about 5 weight percent to
about 50 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment of the
composition, the Gotu kola powder is present at a concentration
from at least about 10 weight percent to about 30 weight percent of
the total dry weight of active ingredients of the composition. In
one embodiment of the composition, the Gotu kola powder extract is
present at a concentration at least about 12 weight percent of the
total dry weight of active ingredients of the composition.
[0259] In one embodiment of the composition, the Ginko biloba leaf
extract is present at a concentration from at least about 5 weight
percent to about 50 weight percent of the total dry weight of
active ingredients of the composition. In one embodiment of the
composition, the Ginko biloba leaf extract is present at a
concentration from at least about 10 weight percent to about 30
weight percent of the total dry weight of active ingredients of the
composition. In one embodiment of the composition, the Ginko biloba
leaf extract is present at a concentration at least about 12 weight
percent of the total dry weight of active ingredients of the
composition.
[0260] In one embodiment of the composition, the Aloe vera powder
is present at a concentration from at least about 5 weight percent
to about 50 weight percent of the total dry weight of active
ingredients of the composition. In one embodiment of the
composition, the Aloe vera powder is present at a concentration
from at least about 10 weight percent to about 30 weight percent of
the total dry weight of active ingredients of the composition. In
one embodiment of the composition, the Aloe vera powder is present
at a concentration of at least about 12 weight percent of the total
dry weight of active ingredients of the composition.
[0261] In some embodiments, the herb-containing compositions of the
invention are formulated to contain at least one excipient such as,
e.g., calcium carbonate; croscarmellose sodium; dicalcium
phosphate; magnesium stearate; microcrystalline cellulose; modified
cellulose; silica; and stearic acid.
EQUIVALENTS
[0262] While the invention has been described in connection with
the specific embodiments thereof, it will be understood that it is
capable of further modification. Furthermore, this application is
intended to cover any variations, uses, or adaptations of the
invention, including such departures from the present disclosure as
come within known or customary practice in the art to which the
invention pertains, and as fall within the scope of the appended
claims.
* * * * *
References